Obesity gene and use thereof

ABSTRACT

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human obesity and diabetes predisposing gene, specifically the TBC1D1 gene, some mutant alleles of which cause susceptibility to obesity and/or diabetes. More specifically, the invention relates to germline mutations in the TBC1D1 gene and their use in the diagnosis of predisposition to obesity and diabetes. Finally, the invention relates to the screening of the TBC1D1 gene for mutations/alterations, which are useful for diagnosing the predisposition to obesity.

CROSS REFERENCE TO OTHER U.S. APPLICATIONS

This application claims the benefit (under 35 U.S.C. § 119(e)) of U.S.Provisional Application Ser. No. 60/407,817 filed Sep. 3, 2002, and U.S.Provisional Application Ser. No. 60/433,074 filed Dec. 13, 2002, thecontents of both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates generally to the field of human genetics, andparticularly to an isolated human obesity predisposing gene and usethereof.

BACKGROUND OF THE INVENTION

Generally, obesity is defined as an excess of adipose tissue; andclinically, it is defined as that amount of adiposity that imparts ahealth risk. Even mild obesity, at 20% over desirable weight accordingto standard height-weight charts, may increase the risk for disease andpremature death. While the etiology of obesity and diabetes is notentirely overlapping, it is now amply clear that both share appreciablebiochemical and physiological components.

The incidence of the metabolic disorders of diabetes and obesity hasreached epidemic levels. It has been estimated that over 120 millionAmericans are clinically over-weight and more than ten million Americansare diagnosed with diabetes every year. Moreover, obesity and diabetescan cause or contribute to the development of, or at least affect thetreatment of, other diseases and disorders such as cardiovasculardiseases, stroke, hypertension, and kidney failure. The combinedeconomic burden of diabetes and obesity and the co-morbiditiesassociated with these disorders is estimated to be over $100 billion ayear. Obesity and diabetes have a major impact on human health and thevarious national healthcare systems all over the world.

Recently launched weight-loss drugs have failed or have demonstratedlimited efficacy and undesirable side effects. Similarly, despite atremendous medical need, the pharmaceutical industry has realized onlylimited success developing therapeutics to manage diabetes. The mostcommon therapeutics (sulfonylureas) are not effective and the mostpromising new drugs (thiazolidinediones) have demonstrated rare butfatal side effects. Thus, there is an urgent need for a morecomprehensive understanding of the molecular basis of obesity anddiabetes, for diagnosis tests that allow early detection ofpredispositions to the disorders, and for more effective pharmaceuticalsfor preventing and treating the diseases without undesirable sideeffects.

SUMMARY OF THE INVENTION

This invention provides the first evidence implicating specificmutations in the TBC1D1 gene (also known as the cg79 gene) withsusceptibility to obesity, thus associating the functions of the TBC1D1gene product with increased adiposity and associated increased healthrisk.

In a first aspect of the invention, novel nucleotide sequences and aminoacid sequences relating to the TBC1D1 gene and protein are provided. Inparticular, a nucleotide sequence encoding the wild-type full-lengthTBC1D1 protein has been discovered. In addition, mutations in the TBC1D1nucleotide sequence associated with an elevated risk of obesity havealso been discovered based on familial linkage analyses. Thus, mutantTBC1D1 nucleotide sequences including such mutations are disclosed, asare altered TBC1D1 amino acid sequences. Additionally, severalheretofore unknown alternatively spliced forms of the TBC1D1 codingsequence (CDS), and corresponding mRNA/cDNA sequences, have beendiscovered, and the sequences of such alternative splice forms aredisclosed.

In a second aspect of the invention, a method for detectingsusceptibility in an individual to obesity and/or diabetes is provided.Thus, the present invention provides methods for determining whether asubject is at risk for developing obesity and/or diabetes due to amutation in the TBC1D1 gene. This method relies on the fact that theinventors have correlated mutations in the TBC1D1 with the diseases. Itwill be understood by those of skill in the art, given the disclosure ofthe invention, that such mutations are associated with a susceptibilityto obesity and/or diabetes, and that a variety of methods may beutilized to detect mutations in the TBC1D1 gene, including the mutationsdisclosed herein, which are associated with a susceptibility to obesityand/or diabetes.

The method can include detecting, in a tissue of the subject, thepresence or absence of a polymorphism of the TBC1D1 gene or a TBC1D1gene product. The detection of a polymorphism in the TBC1D1 gene mayinclude ascertaining the existence of at least one of: a deletion of oneor more nucleotides; an addition of one or more nucleotides; asubstitution of one or more nucleotides; a gross chromosomalrearrangement; an alteration in the level of a messenger RNA transcript;the presence of a non-wild type splicing pattern of a messenger RNAtranscript; a non-wild type level of TBC1D1 protein; and/or an aberrantlevel of TBC1D1 protein.

For example, detecting the polymorphism can include (i) providing aprobe/primer comprised of an oligonucleotide that hybridizes to a senseor antisense sequence of the TBC1D1-encoding nucleic acid, or naturallyoccurring mutants thereof, or 5′ or 3′ flanking sequences naturallyassociated with the TBC1D1 gene; (ii) contacting the probe/primer to anappropriate nucleic acid containing sample; and (iii) detecting, byhybridization of the probe/primer to the nucleic acid, the presence orabsence of the polymorphism; e.g. wherein detecting the polymorphismcomprises utilizing the probe/primer to determine the nucleotidesequence of a TBC1D1 gene and, optionally, of the flanking nucleic acidsequences. For instance, the primer can be employed in a polymerasechain reaction (PCR), in a ligase chain reaction (LCR) or otheramplification reactions known to a skilled artisan. In alternateembodiments, the level of a TBC1D1 protein is detected in an immunoassayusing an antibody that is specifically immunoreactive with the TBC1D1protein.

In a third aspect of the invention, compounds that are agonists orantagonists of a normal (functional) TBC1D1 bioactivity are provided, asare their use in preventing or treating obesity and/or diabetes. Forexample, to ameliorate disease symptoms involving insufficientexpression of a TBC1D1 gene and/or inadequate amount of functionalTBC1D1 bioactivity in a subject, a gene therapeutic (comprising a geneencoding a functional TBC1D1 protein) or a protein therapeutic(comprising a functional TBC1D1 protein or fragment thereof) can beadministered to a subject. Alternatively, agonists or antagonists ofTBC1D1 function (wild-type or mutant) or a TBC1D1 receptor or a receptorfor fragments of TBC1D1 can be administered.

In a fourth aspect of the invention, compounds that are antagonists of adisease causing TBC1D1 bioactivity are provided; as are and their use inpreventing or treating obesity. For example, to ameliorate diseasesymptoms involving expression of a mutant TBC1D1 gene or aberrantexpression of a normal TBC1D1 gene in a subject, a therapeuticallyeffective amount of a small interfering RNA (siRNA), antisense,ribozyme, or triple helix molecule, to reduce or prevent gene expressionmay be administered to the subject. Alternatively, to ameliorate diseasesymptoms involving the regulation via the TBC1D1 protein or TBC1D1protein fragments of an upstream or downstream element in aTBC1D1-mediated biochemical pathway (e.g. signal transduction), atherapeutically effective amount of an agonist or antagonist compound(e.g. small molecule, peptide, peptidomimetic, protein or antibody),which can prevent normal binding of the wild type TBC1D1 protein, caninduce a therapeutic effect.

In another aspect of the invention, assays, e.g., for screening testcompounds to identify antagonists (e.g. inhibitors), or alternatively,agonists (e.g. potentiators), of an interaction between a TBC1D1 proteinand, for example, a protein or nucleic acid that binds to the TBC1D1protein, or fragments of TBC1D1, are provided. An exemplary methodincludes the steps of (i) combining a TBC1D1 polypeptide or bioactivefragments thereof, a TBC1D1 target molecule (such as a TBC1D1 ligand ornucleic acid), and a test compound, e.g., under conditions wherein, butfor the test compound, the TBC1D1 protein and TBC1D1 target molecule areable to interact; and (ii) detecting the formation of a complex whichincludes the TBC1D1 protein and the target molecule either by directlyquantitating the complex or by measuring inductive effects of the TBC1D1protein, or fragments of the TBC1D1 protein. A statistically significantchange, such as a decrease, in the interaction of the TBC1D1 protein andTBC1D1 target molecule in the presence of a test compound (relative towhat is detected in the absence of the test compound) is indicative of amodulation (e.g., inhibition or potentiation of the interaction betweenthe TBC1D1 protein, or fragments of the TBC1D1 protein, and the targetmolecule).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the exon structures of variousalternatively spliced forms of TBC1D1 coding sequence. The sequences ofthe exons identified by numbers in the FIGURE are provided in thesequence listing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery of polymorphismsin the TBC1D1 gene that are genetically linked to obesity. In addition,it has also been found that the TBC1D1 protein (TBC1D1) is involved in adiabetes pathway and may function in cellular glucose uptake. Based onthese findings, the invention provides therapeutic methods, compositionsand diagnostic assays for obesity and/or diabetes based onTBC1D1-encoding nucleic acids, and the TBC1D1 protein.

The inventors have discovered that a number of splice variants of TBC1D1exist, each of which is encoded by a unique combination of exons thatare spliced together to form the TBC1D1 coding sequence (CDS). The mostcommon form of the TBC1D1 CDS is provided as SEQ ID NO:1. Thecorresponding amino acid sequence is set forth in SEQ ID NO:2. Inaddition, mutant cDNAs bearing germline mutations in their CDSs havealso been isolated. The sequences of the CDSs of these mutanttranscripts are shown in SEQ ID NOs:15, 17, 19 and 21, and the aminoacid sequences encoded by the CDSs are shown in SEQ ID NOs:16, 18, 20and 22, respectively. The mutation found in disequilibrium with obesityand provided in SEQ ID NO:15 is C373T, which corresponds to an aminoacid variant R125W, provided in SEQ ID NO:16. The mutation found indisequilibrium with obesity and provided in SEQ ID NO:17 is T683G, whichcorresponds to an amino acid variant V228G, provided in SEQ ID NO:18.The mutation found in disequilibrium with obesity and provided in SEQ IDNO:19 is C1174G, which corresponds to an amino acid variant L392V,provided in SEQ ID NO:20. The mutations found in disequilibrium withobesity and provided in SEQ ID NO:21 are T683G and C1174G, whichcorrespond to amino acid variants V228G and L392V, respectively,provided in SEQ ID NO:22.

Based on cDNA cloning and sequence analysis, a large number of exons ofTBC1D1 have been found. These exons can be alternatively spliced tocreate several different splice variants, as described below. Thenucleotide sequences of the individual exons encoding portions of allknown forms of TBC1D1 are provided in SEQ ID NOs:33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,and 80. Exon 1 encodes a portion of the TBC1D1 transcript 5′-UTR, aswell as the first 139 amino acid residues from the N-terminus of TBC1D1.The portion of the 5′ UTR encoded by exon 1 corresponds to that foundjust upstream (5′) of the translation initiation codon—a portion presentin all known TBC1D1 transcripts.

The structural arrangements of exons in the coding regions of variousalternatively spliced TBC1D1 CDS variants are shown schematically inFIG. 1. Each of the structures depicted may be appended to either of thesequences encoded by exons 22 and 23, as described below, to create acDNA with 5′ and 3′ UTRs. The nucleotide sequences corresponding to theCDSs of structural variants A-P, as illustrated in FIG. 1, are providedin SEQ ID NOs:1, 29, 31, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, and 105, respectively, and their encoded amino acid sequences areprovided in SEQ ID NOs:2, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104 and 106, respectively. TBC1D1 transcripts have been foundto contain one of two alternative 5′-UTRs. The 5′-most regions of thesealternative 5′-UTRs correspond to either of the sequences encoded by oneof two nontranslated exons, designated exon 22 (SEQ ID NO:79) and exon23 (SEQ ID NO:80). To create a full-length cDNA transcript, exon 22 orexon 23 is spliced to the 5′ end of the first coding exon—exon 1 (SEQ IDNO:33)—to form cDNAs containing coding sequences corresponding to any ofthe CDS structures diagramed in FIG. 1. Importantly, exons 22 and 23 areseparated by 3 kilobasepairs in the genomic DNA (SEQ ID NO:28) andtherefore are derived from separate promoters. It is likely that thesepromoters comprise important regulatory elements that impart tissueand/or temporal specificity to the distribution of TBC1D1 transcripts.

The present invention also relates to TBC1D1 agonists and antagonistsand their use in treating obesity and diabetes. For example, (i) nucleicacid molecules encoding functional TBC1D1 protein; (ii) nucleic acidsthat are effective antisense, ribozyme, siRNA and triplex antagonists ofnucleic acids encoding functional TBC1D1 protein; (iii) functionalTBC1D1 proteins or peptides; (iv) anti-TBC1D1 antibodies; (v) smallorganic molecules affecting wild-type or mutant TBC1D1 function orTBC1D1 interaction with a TBC1D1 interactor, and preparations of suchcompositions, are disclosed herein. In addition, the invention providesdrug discovery assays for identifying additional agents that agonize orantagonize the biological function of TBC1D1 protein (e.g. by alteringthe interaction of TBC1D1 molecules with either downstream or upstreamelements in the biochemical (e.g. signal transduction pathway).Moreover, the present invention provides assays for diagnosing whether asubject has a predisposition towards developing obesity and/or diabetes.

Nucleic Acids and Proteins

The terms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, etc.Modifications also include intra-molecular crosslinking and covalentattachment to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by genes may also be included in a polypeptide.

The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide that has been separated from components that accompany it inits natural state. A protein is substantially pure or isolated when atleast about 20% by weight of the total protein content in a compositionis the specified protein. However, the present invention also provides,in preferred embodiments, protein compositions containing a protein ofthe present invention at a content of at least 30%, 40%, 50%, 60%, 70%,80% or 90% of the total proteins in the protein compositions on aweight-to-weight basis. Protein purity or homogeneity may be indicatedby a number of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualizing aparticular polypeptide band upon staining the gel. For certain purposes,higher resolution may be provided by using HPLC, capillaryelectrophoresis, or other means well known in the art which are utilizedfor protein purification.

The term “high stringency hybridization conditions,” when used inconnection with nucleic acid hybridization, means hybridizationconducted overnight at 42° C. in a solution containing 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured and sheared salmon sperm DNA, with hybridization filterswashed in 0.1×SSC at about 65° C. The term “moderate stringencyhybridization conditions,” when used in connection with nucleic acidhybridization, means hybridization conducted overnight at 37 degrees C.in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10%dextran sulfate, and 20 microgram/ml denatured and sheared salmon spermDNA, with hybridization filters washed in 1×SSC at about 50° C. It isnoted that many other hybridization methods, solutions and temperaturescan be used to achieve comparably stringent hybridization conditions aswill be apparent to skilled artisans.

For purposes of comparing two different nucleic acid or polypeptidesequences, one sequence (comparing sequence) may be described to be aspecific “percent identical to” another sequence (reference sequence) inthe present disclosure. In this respect, the percentage identity isdetermined by the algorithm of Karlin and Altschul, Proc. Natl. Acad.Sci. USA, 90:5873-5877 (1993), which is incorporated into the variousBLAST programs. Specifically, the percentage identity is determined bythe “BLAST 2 Sequences” tool, which is available on the world wide webat ncbi.nlm.nih.gov. See Tatusova and Madden, FEMS Microbiol. Lett.,174(2):247-250 (1999). For pairwise DNA-DNA comparison, the BLASTN 2.1.2program is used with default parameters (Match: 1; Mismatch: −2; Opengap: 5 penalties; extension gap: 2 penalties; gap x_dropoff: 50; expect:10; and word size: 11, with filter). For pairwise protein-proteinsequence comparison, the BLASTP 2.1.2 program is employed using defaultparameters (Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff:15; expect: 10.0; and wordsize: 3, with filter).

As used herein, the term “interacting” or “interaction” means that twoprotein domains, fragments, or complete proteins exhibit sufficientphysical affinity to each other so as to bring the two “interacting”protein domains, fragments, or proteins physically close to each other.An extreme case of interaction is the formation of a chemical bond thatresults in continual and stable proximity of the two domains orproteins. Interactions that are based solely on physical affinities,although usually more dynamic than chemically bonded interactions, canbe equally effective in co-localizing two proteins. Examples of physicalaffinities and chemical bonds include, but are not limited to, forcescaused by electrical charge differences, hydrophobicity, hydrogen bonds,Van der Waals forces, ionic forces, covalent linkages, and combinationsthereof. The state of proximity between the interacting domains orentities may be transient or permanent, reversible or irreversible. Inany event, it is in contrast to, and distinguishable from, contactcaused by natural random movement of two entities. Typically althoughnot necessarily, an “interaction” is exhibited by the binding betweenthe interacting domains or entities. Examples of interactions includespecific interactions between antigen and antibody, ligand and receptor,enzyme and substrate, and the like.

As used herein, two proteins or protein fragments are deemed to interactwith each other when an interaction is detected using a yeast two-hybridmethod and/or by co-immunoprecipitation.

Accordingly, the present invention provides isolated TBC1D1 nucleic acidmolecules. The nucleic acid molecules can be in the form of DNA, RNA, ora chimera or hybrid thereof, and can be in any physical structuresincluding single-stranded or double-stranded molecules, or in the formof a triple helices.

In one embodiment, the isolated TBC1D1 nucleic acid molecule has asequence of SEQ ID NO:28, or some fragment, or collection of fragments,thereof. Conveniently, by way of examples, the isolated TBC1D1 nucleicacid molecule in accordance with this embodiment can be prepared byisolating genomic DNA from human cells or tissues, and cloning oramplifying the desired fragment or fragments.

In another embodiment, the isolated TBC1D1 nucleic acid molecule has asequence of SEQ ID NOs:1, 11, 12, 13, 15, 17, 19, 21, 26, 29, 31, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, or the complementor ribonucleic acid equivalent thereof. Conveniently, by way ofexamples, the isolated TBC1D1 nucleic acid molecule in accordance withthis embodiment can be prepared by isolating the TBC1D1 mRNA from humancells or tissues, or by reverse transcribing a TBC1D1 mRNA molecule andamplifying the resulting cDNA molecule.

In yet another embodiment, an isolated nucleic acid molecule is providedwhich has a sequence that is at least 50%, preferably at least 60%, morepreferably at least 75%, 80%, 82%, 85%, even more preferably at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NOs: 1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29, 31,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, or thecomplement or ribonucleic acid equivalent thereof. Preferably, suchnucleic acid molecules encode a polypeptide having the sequence of SEQID NOs:2, 14, 16, 18, 20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, or 106.

In specific embodiments, nucleic acids are provided having at least acontiguous span of at least 2600, 2650, 2800, 3000, 3200 or 3504nucleotides of SEQ ID NOs: 1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29,31, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, or thecomplement or ribonucleic acid equivalent thereof.

As is apparent to skilled artisans, nucleic acids homologous to, ornucleic acids capable of hybridizing with, a nucleic acid of thesequence of SEQ ID NOs: 1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29, 31,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105, or thecomplement or ribonucleic acid equivalent thereof, can be prepared bymanipulating a TBC1D1 nucleic acid molecule having a sequence of SEQ IDNOs: 1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29, 31, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, or 105, or the complement or ribonucleicacid equivalent thereof. For example, various nucleotide substitutions,deletions or insertions can be incorporated into the TBC1D1 nucleic acidmolecule by standard molecular biology techniques. As will be apparentto skilled artisans, such nucleic acids are useful irrespective ofwhether they encode a functional TBC1D1 protein. For example, they canbe used as probes for isolating and/or detecting TBC1D1 nucleic acids.In certain embodiments, nucleic acids homologous to, or nucleic acidscapable of hybridizing with, a nucleic acid of the sequence of SEQ IDNOs: 1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29, 31, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, or 105, encode a polypeptide having one ormore TBC1D1 activities. In one embodiment, the proteins encoded by thenucleic acids contain a phosphotyrosine interacting domain (PID) and/ora TBC domain. In another embodiment, the proteins encoded by the nucleicacids contain one or more amino acid substitutions selected from thegroup consisting of R125W, V228G, and L392V. In a specific embodiment,the proteins contain both V228G and L392V substitutions. In anotherspecific embodiment, the isolated nucleic acid molecules are naturallyoccurring allelic variants of the TBC1D1 gene nucleic acid. For example,such an isolated nucleic acid can have a nucleotide sequence accordingto SEQ ID NOs:13, 15, 17, 19, or 21.

In addition, nucleic acid molecules that encode the TBC1D1 proteinhaving an amino acid sequence of SEQ ID NOs: 2, 14, 16, 18, 20, 22, 27,30, 32, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106, arealso intended to fall within the scope of the present invention. As willbe immediately apparent to a skilled artisan, due to genetic codedegeneracy, such nucleic acid molecules can be designed conveniently bynucleotide substitutions in the wild-type TBC1D1 nucleotide sequence ofSEQ ID NO:1 or TBC1D1 sequences according to SEQ ID NOs:13, 15, 17, 19,21, 29, 31, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, or 105.

In addition, the present invention further encompasses nucleic acidmolecules encoding a protein that has a sequence that is at least 75%,preferably at least 85%, 90%, 91%, 92%, 93%, or 94%, and more preferablyat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNOs: 2, 14, 16, 18, 20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, or 106. Preferably, the homologous protein retainsone or more activities of TBC1D1. More preferably, the homologousprotein contains a phosphotyrosine interacting domain (PID) and/or a TBCdomain, or a fragment thereof. The various nucleic acid molecules may beprovided by chemical synthesis and/or recombinant techniques based on anisolated TBC1D1 nucleic acid molecule having a sequence of SEQ ID NOs:1, 11, 12, 13, 15, 17, 19, 21, 26, 28, 29, 31, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, or 105. In preferred embodiment, the proteinhas a sequence according to SEQ ID NOs: 14, 16, 18, 20, 22, 27, 30, 32,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106.

The present invention also encompasses isolated nucleic acids encoding aTBC1D1 protein or a fragment thereof and having a sequence selected from(1) exons 11, 12, and 13, joined together, in that order, (2) exons 11,33, 34, 12, and 13, joined together, in that order, (3) exons 11, 34,12, and 13, joined together, in that order, (4) exons 11, 12, 36, and13, joined together, in that order, (5) exons 11, 33, 34, 12, 36, and13, joined together, in that order, (6) exons 11, 34, 12, 36 and 13,joined together, in that order, (7) exons 11, 33, 12, 36 and 13, joinedtogether, in that order, (8) exons 11, 33, 12, and 13, joined together,in that order, (9) exons 11a, 12, and 13, joined together, in thatorder, (10) exons 11a, 33, 34, 12, and 13, joined together, in thatorder, (11) exons 11a, 34, 12, and 13, joined together, in that order,(12) exons 11a, 12, 36, and 13, joined together, in that order, (13)exons 11a, 33, 34, 12, 36, and 13, joined together, in that order, (14)exons 11a, 34, 12, 36 and 13, joined together, in that order, (15) exons11a, 33, 12, 36 and 13, joined together, in that order, and (16) exons11a, 33, 12, and 13, joined together, in that order. The full-lengthTBC1D1 protein, or any fragment thereof, comprising any of the sixteencombinations of exons described above also falls within the scope of thepresent invention.

In another embodiment of the present invention, oligonucleotides orTBC1D1 fragments are provided having a contiguous span of at least 18,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 50, 75, 100, 125, 150, 200,250, 300, 350 or 400 nucleotides of the sequence of SEQ ID NOs: 1, 11,12, 13, 15, 17, 19, 21, 26, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 or 80, orthe complement or ribonucleic acid equivalent thereof. In a preferredembodiment, the contiguous span includes, e.g., a sequence according toSEQ ID NO:5 or SEQ ID NO:6. In some other embodiments, the nucleic acidsinclude a sequence according to SEQ ID NOs:3, 7, or 9. Preferably, theoligonucleotides are less than the full length of the sequence of SEQ IDNOs:1, 11, 12, 13, 15, 17, 19, 21, 26, 28, or complement or ribonucleicacid equivalent thereof. More preferably the oligonucleotides are nogreater than 1,200, 1,000, 800, 600, 400, 200, 100, or 50 nucleotides inlength. In a preferred embodiment, the oligonucleotides have a length ofabout 19-25, 26-34, 35-50, or 51-100 nucleotides. In other preferredembodiments, the oligonucleotides selectively hybridize, under moderatestringency hybridization conditions, to a nucleic acid having one, butnot the others, of the sequences according to SEQ ID NOs:13, 15, 17, 19,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 80, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103 and 105. Preferably, in such preferred embodiments, theoligonucleotide includes a contiguous stretch of nucleotides of from 15,16, 17, or 18 to 30, 40, or 50 nucleotides. In specific embodiments,such oligonucleotides can hybridize, under high or moderate stringencyhybridization conditions, to a TBC1D1 nucleic acid having one or moremutations selected from C373T, T683G, C1174G (relative to SEQ ID NO:1)and equivalents thereof, or the complement of such a TBC1D1 nucleicacid, but not to TBC1D1 nucleic acids without such mutations (or thecomplements thereof). The term “equivalent” as used in this and othersimilar contexts means the equivalent nucleotide of C373T, T683G orC1174G, relative to SEQ ID NO:1, in other variant TBC1D1 nucleic acids(e.g., alternative spliced forms), although the exact numberrepresenting the location of the equivalent in the variant TBC1D1nucleic acid may be different.

The present invention further encompasses oligonucleotides that have alength of at least 10, 12, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 50, 75, 100, 125, 150, 200, 250, 300, 350 or 400 nucleotides, andare at least 85%, 90%, 92% or 94%, and more preferably at least 95%,96%, 97%, 98%, or 99% identical to a contiguous span of nucleotides ofthe sequence of SEQ ID NOs:1, 11, 12, 13, 15, 17, 19, 21, 26 or 28, orthe complement or ribonucleic acid equivalent thereof of the samelength. Preferably, the oligonucleotides are no greater than 1,200,1,000, 800, 600, 400, 200, 100, or 50 nucleotides. The oligonucleotidescan have a length of about 12-18, 19-25, 26-34, 35-50, or 51-100nucleotides. In a preferred embodiment, the oligonucleotides have alength of about 12-100, 15-75, 17-50, 21-50, or preferably 25-50nucleotides. In one embodiment, the oligonucleotide is a sequenceencoding a contiguous span of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 18, 20, 22, 25, 30, 35, 50, 75, 100, 125 or 150 amino acidsof SEQ ID NOs:2, 14, 16, 18, 20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, or 106. In another embodiment, theoligonucleotides include a mutant sequence according to SEQ ID NOs:23,24, or 25, or the complement thereof.

As will be apparent to skilled artisans, the various oligonucleotides ofthe present invention are useful as probes for detecting TBC1D1 nucleicacids in cells and tissues. They can also be used as primers forprocedures including the amplification of TBC1D1 nucleic acids, orhomologues thereof, sequencing TBC1D1 nucleic acids, and detection ofmutations in TBC1D1 nucleic acids, or homologues thereof. In addition,the oligonucleotides may be used to encode a fragment, epitope or domainof TBC1D1, or a homologue thereof, which is useful in a variety ofapplications including use as an antigenic epitope for preparingantibodies against TBC1D1.

It should be understood that the nucleic acid molecules of the presentinvention may be standard nucleic acids with conventional nucleotidebases and backbones, but can also be various modified forms of nucleicacids, or analogs thereof, e.g., having therein modified nucleotidebases or backbones.

In another embodiment, a hybrid or chimeric nucleic acid molecule isprovided comprising any one of the above-described nucleic acidmolecules of the present invention covalently linked to a non-TBC1D1nucleic acid. In a specific embodiment, the present invention provides avector comprising an insert of a TBC1D1 nucleic acid. Preferably, thevector is a DNA vector comprising as an insert of any one of theabove-described nucleic acid molecules of the present invention. In aspecific embodiment, the vector is an expression vector. Any suitablevectors may be used for purposes of the present invention. A typicalexpression vector should have a suitable promoter operably linked to aTBC1D1 nucleic acid of the present invention. The vectors of the presentinvention may be used to amplify the nucleic acid molecules of thepresent invention, or to introduce the nucleic acids into host cells.Such vectors may also be used for purposes of producing proteins encodedby the nucleic acids in a cell free system or in cells or tissues. Largeamounts of the nucleic acids of the present invention may be produced byreplication in a suitable host or transgenic animals. Constructsprepared for introduction into a prokaryotic or eukaryotic host maycomprise a replication system recognized by the host, including theintended polynucleotide fragment encoding the desired polypeptide. Suchconstructs will preferably also include transcriptional andtranslational regulatory sequences operably linked to thepolypeptide-encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary functional sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences. Secretion signals may also be included whereappropriate in order to allow the expressed protein to cross and/orlodge in cell membranes, and thus attain its functional topology, or besecreted from the cell. Such vectors may be prepared by means ofstandard recombinant techniques well known in the art.

Thus, the present invention further contemplates host cells into whichany of the nucleic acid molecules of the present invention have beenintroduced from an exogenous source. The nucleic acid molecules of thepresent invention may be introduced into any type of suitable hostcells, including, but not limited to, bacteria, yeast cells, plantcells, insect cells, and animal cells. The nucleic acid molecules of thepresent invention can be introduced exogenously into a host cell by anymethods known in the art. When a nucleic acid molecule of the presentinvention is appropriately incorporated into a suitable expressionvector and introduced into host cells, proteins may be recombinantlyexpressed within the host cells. Accordingly, the present invention alsoprovides methods for recombinantly producing TBC1D1 protein, orfragments or homologues thereof, which includes the steps of introducingan expression vector containing a TBC1D1 nucleic acid molecule into acell and expressing TBC1D1 in the host cell. The proteins expressed inthis manner may be isolated and/or purified by standard purificationtechniques known in the art. Methods for making the host cells andrecombinantly expressing TBC1D1 will be apparent to skilled artisans.Alternatively, an in vitro translation method can also be used inproducing TBC1D1 protein, or homologues, derivatives, or fragmentsthereof. For example, a wheat germ extract system or rabbit reticulocytelysate system may be used for in vitro translation, as will be apparentto a skilled person in the art.

In yet another embodiment of the present invention, a nucleic acidmicrochip or microarray is provided comprising one or more of theforegoing isolated nucleic acid molecules of the present invention. Asis known in the art, with nucleic acid microchips a large number ofnucleic acid molecules can be attached or immobilized in an array on asolid support, e.g., a silicon chip or glass slide. See Lipshutz et al.,Biotechniques, 19:442-447 (1995); Chee et al., Science, 274:610-614(1996); Kozal et al., Nat. Med. 2:753-759 (1996); Hacia et al., Nat.Genet., 14:441-447 (1996); Saiki et al., Proc. Natl. Acad. Sci. USA,86:6230-6234 (1989); Gingeras et al., Genome Res., 8:435-448 (1998). Themicrochip technologies combined with computerized analysis tools allowfor speedy high throughput screening and analysis. Various techniquesfor making and using nucleic acid microchips are known in the art anddisclosed in, e.g., U.S. Pat. No. 5,925,525 to Fodor et al; Wilgenbus etal., J. Mol. Med., 77:761-786 (1999); Graber et al., Curr. Opin.Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet., 14:441-447(1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996); DeRisi et al.,Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet., 14:610-614(1996); Lockhart et al., Nat. Genet., 14:675-680 (1996); Drobyshev etal., Gene, 188:45-52 (1997), all of which are incorporated herein byreference.

In a preferred embodiment, DNA molecules encoding TBC1D1, or a fragmentor homologue thereof, or the complements or RNA equivalents of such DNAmolecules, are included in a microarray of the present invention. Morepreferably, DNA molecules having a sequence according to the sequence ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 12, 13, 15, 17, 19, 21 23, 24, or 25, orthe complement thereof, are incorporated into a microarray of thepresent invention. In specific embodiments, oligonucleotides areincorporated into the microchips having a contiguous stretch of from 15,16, 17, or 18 to 30, 40, or 50 nucleotides. In specific embodiments,such oligonucleotides can hybridize, under high or moderate stringencyhybridization conditions, to a TBC1D1 nucleic acid having one or moremutations selected from C373T, T683G, C1174G (relative to SEQ ID NO:1)and the equivalents or complement thereof, but not to TBC1D1 nucleicacids without such mutations, or the complements thereof.

In accordance with another aspect of the present invention, an isolatedTBC1D1 polypeptide is provided. In one embodiment, the TBC1D1polypeptide comprises the full sequence of SEQ ID NOs: 2, 14, 16, 18,20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,or 106.

Additionally, the present invention also encompasses a polypeptidehaving an amino acid sequence that is at least 50%, preferably at least60%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,and even more preferably at least 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NOs: 2, 14, 16, 18, 20, 22, 27, 30,32, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106. Forexample, polypeptides are provided comprising the sequence of SEQ IDNOs: 2, 14, 16, 18, 20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, or 106 with no more than 1%, 2%, 3%, 4%, 5% or 10%amino acid deletions, insertions or substitutions. Preferably, any aminoacid substitutions are conservative substitutions. Preferably, thehomologous polypeptide retains one or more activities of TBC1D1. Morepreferably, the homologous polypeptide contains a PID domain and/or aTBC domain. In a specific embodiment, the homologous polypeptide is anaturally occurring variant of TBC1D1 identified in a human population.Such a variant may be identified by assaying the TBC1D1 nucleic acids orTBC1D1 protein in a population, as is generally known in the art. Thenucleic acid variant thus identified can be isolated or alternativelyproduced by mutagenesis of the TBC1D1 nucleic acid of the sequence ofSEQ ID NO:1. The TBC1D1 variant can then be prepared by recombinantexpression using the nucleic acid variant as a template fortranscription and translation.

The present invention further encompasses polypeptides having acontiguous span of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400,500, 600, 700, 800, 860, 900, preferably a contiguous span of from 6, 7,8 or 9 to 10, 11, 12, 15 amino acids of the sequence of SEQ ID NOs: 2,14, 16, 18, 20, 22, 27, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, or 106. For example, TBC1D1 fragments can be generated as aresult of the deletion of a contiguous span of a certain number of aminoacids from either or both of the amino and carboxyl termini of theTBC1D1 protein having the sequence of SEQ ID NOs: 2, 14, 16, 18, 20, 22,27, 30, 32, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106.In specific embodiments, the TBC1D1 fragments contain immunogenic orantigenic epitopes. Such epitopes can be readily predicted by computerprograms such as MacVector from International Biotechnologies, Inc. andProtean/DNAStar from LaserGene, Inc. In addition, epitopes can also beselected experimentally by any methods known in the art, e.g., in U.S.Pat. Nos. 4,833,092 and 5,194,392, both of which are incorporated hereinby reference.

In preferred embodiments, the present invention provides peptidescomprising or consisting of a contiguous span of from 5, 6, 7, 8 or 9 to10, 11, 12, 15, 20 amino acids of SEQ ID NO:16, having the amino acidsubstitution R125W; a contiguous span of from 5, 6, 7, 8 or 9 to 10, 11,12, 15, 20 amino acids of SEQ ID NO:18, having the amino acidsubstitution V228G; and a contiguous span of from 5, 6, 7, 8 or 9 to 10,11, 12, 15, 20 amino acids of SEQ ID NO:20, having the amino acidsubstitution L392V. Such peptides can be used as antigens to produceantibodies specific to mutant TBC1D1 proteins harboring such amino acidsubstitutions.

In addition, the present invention is also directed to polypeptides thatare homologous to the foregoing TBC1D1 fragments. Such a homologouspolypeptide may have the same length as one of the foregoing TBC1D1fragments of the present invention (e.g., from 5 to 50, from 5 to 30, orfrom 7 to 25, or preferably 8 to 20 amino acids) but has an amino acidsequence that is at least 75%, 80%, 85%, 90%, preferably at least 95%,96%, 97%, 98%, or, more preferably, at least 99% identical to the aminoacid sequence of the corresponding TBC1D1 fragment. For example,polypeptides are provided with no more than 1%, 2%, 3%, 4%, 5% or 10%amino acid deletions, insertions or substitutions to the above-describedTBC1D1 fragments. Preferably, any amino acid substitutions areconservative substitutions.

The protein fragments of the present invention may still retain thebiological functions of TBC1D1 or one or more activities of TBC1D1. Forexample, such protein fragments may be immunogenic and thus can be usedin producing antibodies against TBC1D1. The protein fragments may alsobe antigenic and thus can bind to an antibody specific against TBC1D1.In addition, where a protein fragment of the present invention lacks oneor more TBC1D1 activities, it can be used as a competitive inhibitor ofTBC1D1 activities by specifically competing with TBC1D1 protein forbinding partners.

Additionally, the present invention further relates to a hybrid orchimeric polypeptide having any one of the foregoing polypeptides of thepresent invention covalently linked to another polypeptide. Such otherpolypeptides can also be one of the foregoing polypeptides of thepresent invention. Alternatively, such other polypeptides are not one ofthe foregoing polypeptides of the present invention. Preferably, suchother polypeptides are non-TBC1D1 polypeptides.

Methods of Use Diagnosis

Proof that any particular gene located within a genetically definedinterval is a disease susceptability locus is obtained by findingsequences in DNA or RNA extracted from affected kindred members thatcreate abnormal gene products or abnormal levels of gene product. Suchdisease susceptibility alleles will co-segregate with the disease inlarge kindreds. In identifying a disease susceptability locus, the keyis to find polymorphisms or mutations that are serious enough to causean obvious disruption to the normal function of the gene product. Thesemutations can take a number of forms. The most severe forms would beframe-shift mutations or large deletions, which would cause the gene tocode for an abnormal protein or would significantly alter proteinexpression. Less severe disruptive mutations would include smallin-frame deletions and base pair substitutions which result innonconservative amino acid substitutions in the encoded protein thatwould have a significant effect on the protein produced; such as changesto or from a cysteine residue, from a basic residue to an acidic residueor vice versa, from a hydrophobic to hydrophilic residue or vice versa,or other mutations which would affect secondary, tertiary or quaternaryprotein structure. Small deletions or base pair substitutions can alsosignificantly alter protein expression by changing transcription levels,exon splicing patterns, mRNA stability, or translational efficiency ofthe gene transcript. Silent mutations—mutations resulting inconservative amino acid substitutions—would not generally be expected todisrupt protein function. Causal mutations that co-segregate with thedisease phenotype can also be found in the promoter of the gene. Thesemutations can interfere with the binding of regulatory factors and inthis way alter the transcription of the gene and therefore impact thefunction of the gene.

In one aspect, the invention features probes and primers for use in aprognostic or diagnostic assay. For instance, the present invention alsoprovides a probe/primer comprising a substantially purifiedoligonucleotide, which oligonucleotide comprises a region of nucleotidesequence that hybridizes under high or medium stringent hybridizationconditions or physiological conditions to at least approximately 12, 15,17, 18, 19, 20, 21, 22, 23, 24, preferably 25, 26, 27, 28, 29, 30, morepreferably 40, 50 or 75 consecutive nucleotides of sense or anti-sensesequence of TBC1D1, including 5′ and/or 3′ untranslated regions.Examples of TBC1D1 sequences from which such probes or primers can bederived include those according to SEQ ID NOs:1, 3, 5, 6, 7, 9, 11, 12,13, 15, 17, 19, 21, 23, 24, 25, 26, 28, 29, 31, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103 or 105. In certain embodiments, theprobe/primer comprises a contiguous stretch of 12, 13, 14, 15, 16, 17 to18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 or 70nucleotides of SEQ ID NOs:15, 17, 19, and 21, and is capable ofselectively hybrizing with a nucleic acid having the sequence of SEQ IDNOs:15, 17, 19, and 21, respectively, under high or moderate stringencyhybridization conditions, or physiological conditions. That is, theprobe/primer does not hybridize to a nucleic acid having a consensuswild-type nucleotide sequence, but hybridize to the mutant sequencesdiscovered in obesity patients according to the present invention. Inpreferred embodiments, the probes/primers can have a contiguous stretchof 12, 13, 14, 15, 16, 17 to 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 50, 60 or 70 nucleotides of SEQ ID NOs:15, 17, 19, and 21,which spans one or more of the mutations according to the presentinvention, i.e., C373T, T683G, and/or C1174G. In other embodiments, theprobes/primers can have a contiguous stretch of at least 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60or 70 nucleotides of SEQ ID NO:26, which spans one or more of themutations according to the present invention, i.e., C508G, T818G, and/orC1309G. For example, such selective hybridization probes can have asequence according to SEQ ID NO:23, 24 or 25. In preferred embodiments,the probe further comprises a label group attached thereto and able tobe detected, e.g. the label group is selected from amongstradioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

In a further aspect, the present invention features methods fordetermining whether a subject is at risk for developing obesity and/ordiabetes. According to the diagnostic and prognostic methods of thepresent invention, alteration of the wild-type TBC1D1 locus is detected.“Alteration of a wild-type gene” encompasses all forms of mutationsincluding deletions, insertions and point mutations in the coding andnoncoding regions. Deletions may be of the entire gene or of only aportion of the gene. Point mutations may result in stop codons,frameshift mutations or amino acid substitutions. Point mutations ordeletions in the promoter can change transcription and thereby impactgene function. Somatic mutations are those that occur only in certaintissues and are not inherited in the germline. Germline mutations can befound in any of a body's tissues and are inherited. The finding ofTBC1D1 germline mutations thus provides diagnostic information. A TBC1D1allele that is not deleted (e.g., is found on the sister chromosome to achromosome carrying a TBC1D1 deletion) can be screened for othermutations, such as insertions, small deletions, substitutions and otherpoint mutations. Events generating point mutations may occur inregulatory regions such as in the promoter of the gene, or in intronicregions, or at intron/exon junctions.

In one embodiment, the diagnostic/prognostic method includes a step ofdetermining the presence or absence of an amino acid substitution,deletion, insertion, or protein truncation in the phosphotyrosineinteracting domain (PID) of TBC1D1 protein of a patient, wherein thepresence of such mutations would indicate an increased likelihood of, orpredisposition to, obesity and/or diabetes. Such amino acid or proteinchanges can be detected at either the nucleotide sequence level or theamino acid sequence level, as will be apparent to ordinarily skilledartisans apprised of the present disclosure. Preferably, thediagnostic/prognostic method includes a step of determining the presenceor absence of an amino acid substitution, deletion, insertion, orprotein truncation in the phosphotyrosine interacting domain (PID) orthe TBC domain of TBC1D1 protein that would affect the structure of theprotein domains. The general structural features of PID domains areknown in the art, and the important amino acid residues or sequences inthe PID domain of TBC1D1 are readily recognizable. For example, theamino acid residues Arg125, Lys119, Ser112, Ser18, and Ser28 (relativeto SEQ ID NO:2) can be critical to the PID domain's optimal interactionwith phosphotyrosine. Non-conserved substitutions (e.g., charged tonon-charged, positively charged to negatively charged, hydrophilic tohydrophobic, and vice versa) of any of these amino acids would indicatean increased likelihood of predisposition or diagnosis of obesity and/ordiabetes. Amino acid changes that affect the TBC1D1 PID domain's bindingof phosphotyrosine can also be readily identified without undueexperimentation by, e.g., generating such changes in the TBC1D1 PIDdomain and testing its ability to bind phosphotyrosine or aphosphotyrosine-containing peptide (e.g., a peptide having the “NPxPY”consensus sequence).

In a specific embodiment, the diagnostic/prognostic method includes astep of determining the presence or absence of an R125W amino acidsubstitution in the TBC1D1 protein of a patient, wherein the presence ofsuch a substitution would indicate an increased likelihood of, orpredisposition to, obesity and/or diabetes. In a preferred embodiment,the presence or absence of a C373T nucleotide substitution isdetermined.

In another specific embodiment, the diagnostic/prognostic methodincludes a step of determining the presence or absence of a V228G aminoacid substitution in the TBC1D1 protein of a patient, wherein thepresence of such a substitution would indicate an increased likelihoodof, or predisposition to, obesity and/or diabetes. In a preferredembodiment, the presence or absence of a T683G nucleotide substitutionis determined.

In another specific embodiment, the diagnostic/prognostic methodincludes a step of determining the presence or absence of an L392V aminoacid substitution in the TBC1D1 protein of a patient, wherein thepresence of such a substitution would indicate an increased likelihoodof, or predisposition to, obesity and/or diabetes. In a preferredembodiment, the presence or absence of a C1174G nucleotide substitutionis determined.

In another preferred embodiment, the presence or absence of both V228Gand L392V amino acid substitutions are determined, wherein the presenceof both substitutions would indicate an increased likelihood of, orpredisposition to, obesity and/or diabetes. In a preferred embodiment,the presence or absence of a T683G-C1174G haplotype is determined,wherein the presence of the T683G-C1174G haplotype would indicate anincreased likelihood of, or predisposition to, obesity and/or diabetes.

Useful diagnostic techniques include, but are not limited to fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE analysis,Southern blot analysis, single stranded conformation analysis (SSCA),RNase protection assay, allele-specific oligonucleotide (ASO)hybridization, allele-specific amplification, dot blot analysis andPCR-SSCP, as discussed in detail further below. Also useful is therecently developed technique of DNA microchip technology. In addition tothe techniques described herein, similar and other useful techniques arealso described in U.S. Pat. Nos. 5,837,492 and 5,800,998, each of whichare incorporated herein by reference.

Predisposition to disease can be ascertained by testing any tissue of ahuman for mutations of the TBC1D1 gene. For example, a person who hasinherited a germline TBC1D1 mutation would be prone to develop obesityand/or diabetes. This can be determined by testing DNA from any tissueof the person's body. Most simply, blood can be drawn and DNA extractedfrom the cells of the blood. In addition, prenatal diagnosis can beaccomplished by testing fetal cells, placental cells or amniotic cellsfor mutations of the TBC1D1 gene. Alteration of a wild-type TBC1D1allele, whether, for example, by point mutation or deletion, can bedetected by any of the means discussed herein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCA) (Orita etal., 1989). This method does not detect all sequence changes, especiallyif the DNA fragment size is greater than 200 bp, but can be optimized todetect most DNA sequence variation. The reduced detection sensitivity isa disadvantage, but the increased throughput possible with SSCA makes itan attractive, viable alternative to direct sequencing for mutationdetection on a research basis. The fragments that have shiftedmotilities on SSCA gels are then sequenced to determine the exact natureof the DNA sequence variation. Other approaches based on the detectionof mismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE) (Sheffield et al., 1991),heteroduplex analysis (HA) (White et al., 1992) and chemical mismatchcleavage (CMC) (Grompe et al., 1989). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation that affects transcription ortranslation of the protein. Other methods that might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A discussion of currently available methodsof detecting DNA sequence variation can be found in a review by Grompe(1993). Once a mutation is known, an allele specific detection approachsuch as allele-specific oligonucleotide (ASO) hybridization, orallele-specific amplification, can be utilized to rapidly screen largenumbers of other samples for that same mutation.

Detection of point mutations may be accomplished by molecular cloning ofthe TBC1D1 allele(s) and sequencing the allele(s) using techniques wellknown in the art. Alternatively, the gene sequences can be amplifieddirectly from a genomic DNA preparation from the tissue, using knowntechniques. The DNA sequence of the amplified sequences can then bedetermined.

There are at least six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single-stranded conformation analysis (SSCA) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Rano and Kidd, 1989). Forallele-specific PCR, primers are used which only hybridize at their 3′ends to templates bearing a particular TBC1D1 mutation. If theparticular TBC1D1 mutation is not present, an amplification product isnot observed. Amplification Refractory Mutation System (ARMS), asdisclosed in European Patent Application Publication No. 0332435 and inNewton et al., 1989, can also be used. Insertions and deletions ofportions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism(RFLP) probes for the gene or surrounding marker genes can be used toscore an alteration of an allele or an insertion in a polymorphicfragment. Such methods are particularly useful for screening relativesof an affected individual for the presence of the TBC1D1 mutation foundin that individual. Other techniques for detecting insertions anddeletions as known in the art can be used.

In the first three methods (SSCA, DGGE and RNase protection assay), anew electrophoretic band appears when a mutation is present. SSCAdetects a band which migrates differentially because the sequence changecauses a difference in single-strand, intramolecular base pairing. RNaseprotection involves cleavage of the mutant polynucleotide into two ormore smaller fragments. DGGE detects differences in migration rates ofmutant sequences compared to wild-type sequences, using a denaturinggradient gel. In an allele-specific oligonucleotide assay, anoligonucleotide is designed which detects a specific sequence, and theassay is performed by detecting the presence or absence of ahybridization signal. In the mutS assay, the mutS protein binds only tosequences that contain a nucleotide mismatch in a heteroduplex betweenmutant and wild-type sequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are generallyless sensitive than sequencing, they are simpler to perform on a largenumber of samples. An example of a mismatch cleavage technique is theRNase protection method. In the practice of the present invention, themethod involves the use of a labeled riboprobe, which is complementaryto the human wild-type TBC1D1 gene coding sequence. The riboprobe andeither mRNA or DNA isolated from the tumor tissue are annealed(hybridized) together and subsequently digested with the enzyme RNase A,which is able to detect some mismatches in a duplex RNA structure. If amismatch is detected by RNase A, it cleaves at the site of the mismatch.Thus, when the annealed RNA preparation is separated on anelectrophoretic gel matrix, if a mismatch has been detected and cleavedby RNase A, an RNA product will be seen which is smaller than the fulllength duplex RNA for the riboprobe and the mRNA or DNA. The riboprobeneed not be the full length of the TBC1D1 mRNA or gene but can be asegment of either. If the riboprobe comprises only a segment of theTBC1D1 mRNA or gene, it is desirable to use a number of these probes toscreen the whole mRNA sequence for mismatches.

In a similar fashion, DNA probes can be used to detect mismatches,through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988;Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches canbe detected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA that mightcontain a mutation can be amplified using PCR before hybridization.Changes in DNA of the TBC1D1 gene can also be detected using Southernhybridization, especially if the changes are gross rearrangements, suchas deletions, insertions and inversions.

DNA sequences of the TBC1D1 gene, which have been amplified by use ofPCR, may also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the TBC1D1gene sequence harboring a known mutation. For example, one oligomer maybe about 30 nucleotides in length (although shorter and longer oligomersare also usable, as well recognized by those of skill in the art),corresponding to a portion of the TBC1D1 gene sequence. By use of abattery of such allele-specific probes, PCR amplification products canbe screened to identify the presence of a previously identified mutationin the TBC1D1 gene. Hybridization of allele-specific probes withamplified TBC1D1 sequences can be performed, for example, on a nylonfilter. Hybridization to a particular probe under high stringencyhybridization conditions indicates the presence of the same mutation inthe nucleic acids of the sample as in the allele-specific probe.

The newly developed technique of nucleic acid analysis via microchiptechnology is also applicable to the present invention. In thistechnique, literally thousands of distinct oligonucleotide probes arebuilt up in an array on a silicon chip. The nucleic acid to be analyzedis fluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using such techniques one can determine thepresence of mutations or even sequence the nucleic acid being analyzed,or one can measure expression levels of a gene of interest. Advantagesof the method include the parallel processing of many, even thousands,of probes at once, which can tremendously increase the rate of analysisand sample throughput. Microchip technologies have been described inpublications by Hacia et al., 1996; Shoemaker et al., 1996; Chee et al.,1996; Lockhart et al., 1996; DeRisi et al., 1996; Lipshutz et al., 1995.Microchip-based methods have already been used to screen humans formutations in the breast cancer gene BRCA1 (Hacia et al., 1996), and thetechnology has been reviewed in a news article in Chemical andEngineering News (Borman, 1996) and has been the subject of an editorial(Nature Genetics, 1996). Also see Fodor (1997).

The most definitive test for mutations in a candidate locus is todirectly compare genomic TBC1D1 sequences from disease patients withthose from a control population. Alternatively, one could sequence mRNAafter reverse transcription and amplification, e.g., by RT-PCR, therebyeliminating the necessity of determining the exon structure of thecandidate gene.

Mutations in disease patients falling outside the TBC1D1 CDS of can bedetected by examining the non-coding regions, within and around theTBC1D1 gene. Such mutations can occur within introns, promoter regions,and regulatory sequences within or near the TBC1D1 gene. An earlyindication that mutations in noncoding regions are important may comefrom Northern blot experiments that reveal mRNA molecules of abnormalsize or abundance in disease patients as compared to controlindividuals. In one embodiment, an intron/exon junction region of aTBC1D1 gene is analyzed, e.g., by PCR amplification followed bysequencing, to determine the presence or absence of a mutation thatresults in altered mRNA splicing, which in turn causes a substantialchange in the encoded TBC1D1 protein. Examples of such a substantialchange include, e.g., premature translation termination, codonframeshift, large deletions of amino acid sequences, etc. Since theconsensus intron/exon junction sequences are well known in the art,mutations in an intron/exon junction of a subject TBC1D1 gene can bereadily identified by comparison and the resulting consequences of themutations can be recognized.

Alteration of TBC1D1 mRNA expression can be detected by any techniquesknown in the art. These include Northern blot analysis, quantitativeRT-PCR and RNase protection. Diminished or increased mRNA expressionindicates an alteration of the wild-type TBC1D1 gene. Alterations ofwild-type TBC1D1 genes can also be detected by screening for alterationsof wild-type TBC1D1 proteins. For example, monoclonal antibodiesimmunoreactive with TBC1D1 can be used to screen a tissue. Lack of aparticular cognate antigen would indicate a TBC1D1 mutation. Antibodiesspecific for products of mutant alleles could also be used to detectmutant TBC1D1 gene products. Such immunological assays can be done inany convenient formats known in the art. These include Western blots,immunohistochemical assays, and enzyme linked immunosorbant assays(ELISAs) or enzyme linked immunofiltration assays (ELIFAs). Any meansfor detecting an altered TBC1D1 protein can be used to detect alterationof wild-type TBC1D1 genes. Functional assays, such as protein bindingdeterminations, can be used. In addition, assays can be used whichdetect TBC1D1 biochemical function. Finding a mutant TBC1D1 gene productindicates alteration of the wild-type TBC1D1 gene.

Allele-specific primers can also be used to identify specific mutantalleles of the TBC1D1 gene. Such primers anneal only to particularTBC1D1 mutant alleles, and thus will only amplify a product when themutant allele is present as a template.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting the pointmutations discussed above. The probes can also be used to detect PCRamplification products. They may also be used to detect mismatches withthe TBC1D1 gene, mRNA or cDNA using other techniques.

It has been discovered that individuals with the wild-type TBC1D1 genedo not have obesity that results from the TBC1D1 allele. However,mutations that affect the function of the TBC1D1 protein are involved inthe susceptability to obesity as shown herein. Thus, the presence of analtered (or a mutant) TBC1D1 gene, which produces a protein having analtered function, directly correlates to an increased risk of disease.In order to detect a TBC1D1 gene mutation, a biological sample isprepared and analyzed for a difference between the sequence of theTBC1D1 allele being analyzed and the sequence of the wild-type TBC1D1allele. Mutant TBC1D1 alleles can be initially identified by any of thetechniques described above. The mutant alleles are then sequenced toidentify or confirm the specific mutation of the particular mutantallele. Alternatively, mutant TBC1D1 alleles can be initially identifiedby identifying mutant (altered) TBC1D1 proteins, using conventionaltechniques, e.g., protein truncation test. cDNA or genomic DNA can thenbe sequenced to identify the specific mutation responsible for themutant protein. The mutations, especially those that lead to an alteredfunction of the TBC1D1 protein, are used for the diagnostic methods ofthe present invention.

The present invention employs definitions and nomenclature commonly usedin the art with specific reference to the gene described in the presentapplication. Such definitions can be found in U.S. Pat. Nos. 5,837,492;5,800,998; 6,261,801; 6,274,720 and 6,274,376, and in Antonarakis et al.(Human Mutation 11: 1-3 (1998)), each of which are incorporated hereinby reference. Such definitions are employed herein unless the contextindicates otherwise.

In order to detect the presence of a TBC1D1 allele predisposing anindividual to obesity and/or diabetes, a biological sample such as bloodis prepared and analyzed for the presence or absence of predisposingalleles of TBC1D1. Results of these tests and interpretive informationare returned to the health care provider for communication to the testedindividual. The results can be transformed into a data set includingdata and information defining the identity or characteristics of theTBC1D1 gene of the tested individual. The data set may includeinformation relating to the nucleotide sequence of the TBC1D1 geneand/or amino acid sequence encoded by the gene, or relative TBC1D1 mRNAor protein expression levels. Alternatively, the data set may simplyinclude alterations in the TBC1D1 gene or protein, or an indication ofthe presence or absence of any disease-predisposing mutations, andoptionally, a description of the specific disease-predisposingmutation(s). Examples of specific predisposing mutations are describedabove. The data or information can be cast in a transmittable form thatcan be communicated or transmitted to another clinical laboratory,physicians or health care providers, or directly to patients. Such atransmittable form can vary and can be of tangible manufactures. Forexample, the data set can be embodied in texts, tables, diagrams,photographs, charts, images or any other visual forms. The data orinformation can be recorded on a tangible medium such as paper orembodied in computer-readable forms (e.g., electronic, electromagnetic,optical or other signals) by computer readable program codes. The datain a computer-readable form can be stored in a computer usable storagemedium (e.g., floppy disks, magnetic tapes, optical disks, and the like)or transmitted directly through a communication infrastructure. Inparticular, the data embodied in electronic signals can be transmittedin the form of e-mail or posted on a secure access website on theInternet or an Intranet. In addition, the information or data can alsobe recorded in an audible form and transmitted through any suitablemedia, e.g., analog or digital cable lines, fiber optic cables, etc.,via telephone, facsimile, wireless mobile phone, Internet phone and thelike.

Diagnoses may be performed by diagnostic laboratories, or,alternatively, diagnostic kits are manufactured and sold to health careproviders or to private individuals for self-diagnosis. Diagnostic orprognostic tests can be performed as described herein or usingwell-known techniques, such as described in U.S. Pat. No. 5,800,998,incorporated herein by reference.

Initially, the screening method involves amplification of the relevantTBC1D1 sequences by the Polymerase Chain Reaction (PCR). In anotherpreferred embodiment of the invention, the screening method involves anon-PCR based strategy. Such screening methods include two-step labelamplification methodologies that are well known in the art. Both PCR andnon-PCR based screening strategies can detect target sequences with ahigh level of sensitivity.

The most popular methods used today generally involve targetamplification. Here, the target nucleic acid sequence is amplified withpolymerases. One particularly preferred method using polymerase-drivenamplification is PCR. PCR and other polymerase-driven amplificationassays can achieve over a million-fold increase in copy number throughthe use of polymerase-driven amplification cycles. Once amplified, theresulting nucleic acid can be sequenced or used as a substrate for DNAprobes.

When the probes are used to detect the presence of the target sequencesor specific mutant alleles of those target sequences (for example, inscreening for obesity susceptibility), the biological sample to beanalyzed, such as blood or serum, may be treated, if desired, to extractthe nucleic acids. The sample nucleic acid may be prepared in variousways to facilitate detection of the target sequence; e.g. denaturation,restriction digestion, electrophoresis or dot blotting. The targetedregion of the analyte nucleic acid usually must be at least partiallysingle-stranded to form hybrids with the targeting sequence of theprobe. If the sequence is naturally single-stranded, denaturation willnot be required. However, if the sequence is double-stranded, thesequence will probably need to be denatured. Denaturation can be carriedout by various techniques known in the art.

Analyte nucleic acid and probe are incubated under conditions thatpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probes,which is used to bind to the analyte, can be made completelycomplementary to the targeted region of human chromosome 4. Therefore,high stringency conditions are desirable in order to prevent falsepositives. However, conditions of high stringency are used only if theprobes are complementary to regions of the chromosome that are unique inthe genome. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, base composition, probe length, andconcentration of formamide. These factors are outlined in, for example,Maniatis et al., 1982 and Sambrook et al., 1989. Under certaincircumstances, the formation of higher order hybrids, such as triplexes,quadraplexes, etc., may be desired to provide the means of detectingtarget sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand that is labeled,either directly or indirectly. Suitable labels, and methods for labelingprobes and ligands are known in the art, and include, for example,radioactive labels which may be incorporated by known methods (e.g.,nick translation, random priming or phosphorylation by kinases), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of these variations are reviewed in, e.g., Matthews andKricka, 1988; Landegren et al., 1988; Mittlin, 1989; U.S. Pat. No.4,868,105, and in EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated inthis invention. This procedure calls for the hybridization of a nucleicacid probe (or an analog such as a methyl phosphonate backbone replacingthe normal phosphodiester), to the low level DNA target. This probe mayhave an enzyme covalently linked to it, such that the covalent linkagedoes not interfere with the specificity of hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe-enzyme-conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes see Jablonski et al., 1986.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding a wild type TBC1D1 allele. Mutant allele specificprobes are also contemplated within the scope of this example andexemplary mutant allele specific probes include probes encompassing thepredisposing or potentially predisposing mutations summarized herein.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate that reacts with a chemiluminescent substrate and produces aluminescent signal. For methods for labeling nucleic acid probesaccording to this embodiment see Martin et al., 1990. Alternatively,hybridization may be detected by an antibody-alkaline phosphataseconjugate that reacts with a chemifluorescent substrate which, whencleaved, generates a fluorescent dye that is detected by fluorescencescanning. In another example, the small ligand is recognized by a secondligand-enzyme conjugate that is capable of specifically complexing tothe first ligand. A well-known embodiment of this example is thebiotin-avidin type of interactions. For methods for labeling nucleicacid probes and their use in biotin-avidin based assays see Rigby etal., 1977 and Nguyen et al., 1992.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting wild type alleles of TBC1D1.Thus, in one example, to detect the presence of wild type alleles ofTBC1D1 in a cell sample, more than one probe complementary to TBC1D1 isemployed and in particular the number of different probes isalternatively 2, 3, or 5 different nucleic acid probe sequences. Inanother example, to detect the presence of mutations in the TBC1D1 genesequence in a patient, more than one probe complementary to TBC1D1 isemployed where the cocktail includes probes capable of binding to theallele-specific mutations identified in populations of patients withalterations in TBC1D1. In this embodiment, any number of probes can beused, and will preferably include probes corresponding to the major genemutations identified as predisposing an individual to obesity. Somecandidate probes contemplated within the scope of the invention includeprobes that include the allele-specific mutations identified herein andthose that have the TBC1D1 regions corresponding to SEQ ID NOs:13, 15,17, 19 and 21, both 5′ and 3′ to the mutation site.

Susceptibility to obesity and/or diabetes can also be detected on thebasis of the alteration of wild-type TBC1D1 polypeptide. Peptidediagnostic or prognostic tests can be performed as described herein orusing well-known techniques, such as described in U.S. Pat. No.5,800,998, incorporated herein by reference. For example, suchalterations can be determined by amino acid sequence analysis inaccordance with conventional techniques. More preferably, antibodies(polyclonal or monoclonal) are used to detect differences in, or theabsence of, TBC1D1 peptides. The antibodies may be prepared inaccordance with conventional techniques. Other techniques for raisingand purifying antibodies are well known in the art and any suchtechniques may be chosen to achieve the preparations claimed in thisinvention. In a preferred embodiment of the invention, antibodies willimmunoprecipitate TBC1D1 proteins, or fragments of the TBC1D1 protein,from solution, as well as react with TBC1D1 peptides on Western orimmunoblots of polyacrylamide gels. In another preferred embodiment,antibodies will detect TBC1D1 proteins and protein fragments in paraffinor frozen tissue sections, using immunocytochemical techniques.

Preferred embodiments relating to methods for detecting TBC1D1 or mutantforms thereof include ELISA, ELIFA, radioimmunoassays (RIA),immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),including sandwich assays using monoclonal and/or polyclonal antibodies.Exemplary sandwich assays are described by David et al. in U.S. Pat.Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.

Alteration of TBC1D1 expression levels may also be detected usingantibodies or other methods described above.

In specific embodiments, the presence or absence of amino acidsubstitutions R125W, V228G, or L392V is detected, either individually orcollectively. In certain preferred embodiments, antibodies may be usedto detect and quantitate mutant forms of TBC1D1.

The present invention also provides a kit for predicting, in anindividual, the effective response to antiobesity drugs. The kit mayinclude a carrier for the various components of the kit. The carrier canbe a container or support, in the form of, e.g., bag, box, tube, orrack, which is optionally compartmentalized. The carrier may define anenclosed confinement for safety purposes during shipment and storage.The kit also includes various components useful in detecting thenucleotide or amino acid variants discovered in accordance with thepresent invention using any of the above-discussed detection techniques.

In one preferred embodiment, the detection kit includes one or moreoligonucleotides useful in detecting the C373T, T683G, and/or C1174Ggenetic variants in the TBC1D1 gene sequence. Preferably, theoligonucleotides are designed such that they hybridize only to a TBC1D1gene sequence containing the particular variants discovered inaccordance with the present invention, under high or moderate stringencyconditions. Thus, the oligonucleotides can be used in mutation-detectingtechniques such as allele-specific oligonucleotides (ASO),allele-specific PCR, TaqMan, chemiluminescence-based techniques,molecular beacons, and improvements or derivatives thereof, e.g.,microchip technologies. The oligonucleotides in this embodimentpreferably have a nucleotide sequence that matches a nucleotide sequenceof the mutant TBC1D1 gene allele containing the specific genetic variantto be detected. The nucleotide variant preferably is not located at the5′ or 3′ end, but in other positions in the oligonucleotides. The lengthof the oligonucleotides in accordance with this embodiment of theinvention can vary depending on its nucleotide sequence and thehybridization conditions employed in the detection procedure.Preferably, the oligonucleotides contain from about 10 nucleotides toabout 100 nucleotides, more preferably from about 15 to about 75nucleotides. Under certain conditions, a length of 18 to 30 may beoptimum. In any event, the oligonucleotides should be designed such thatthey can be used in distinguishing one genetic variant from another at aparticular locus under predetermined stringent hybridization conditions.The hybridization of an oligonucleotide with a nucleic acid and theoptimization of the length and hybridization conditions should beapparent to a person of skill in the art. See generally, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989. Notably, theoligonucleotides in accordance with this embodiment are also useful inmismatch-based detection techniques described above, such aselectrophoretic mobility shift assay, RNase protection assay, mutSassay, etc.

In another embodiment of this invention, the kit includes one or moreoligonucleotides suitable for use in detecting techniques such as ARMS,oligonucleotide ligation assay (OLA), and the like. The oligonucleotidesin this embodiment include a TBC1D1 gene sequence immediately 5′upstream from the genetic variant to be analyzed. The 3′ end nucleotideis a nucleotide variant in accordance with this invention.

The oligonucleotides in the detection kit can be labeled with anysuitable detection marker including but not limited to, radioactiveisotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase),enzyme substrates, ligands and antibodies, etc. See Jablonski et al.,Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques,13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977).Alternatively, the oligonucleotides included in the kit are not labeled,and instead, one or more markers are provided in the kit so that usersmay label the oligonucleotides at the time of use.

In another embodiment of the invention, the detection kit contains oneor more idiotype-specific antibodies, i.e., antibodies that onlyrecognize certain TBC1D1 proteins or polypeptides containing one or moreamino acid substitutions of R125W, V228G and L392V. Methods forproducing and using such antibodies should be apparent to skilledartisans.

Various other components useful in the detection techniques may also beincluded in the detection kit of this invention. Examples of suchcomponents include, but are not limited to, Taq polymerase,deoxyribonucleotides, dideoxyribonucleotides other primers suitable forthe amplification of a target DNA sequence, RNase A, mutS protein, andthe like. In addition, the detection kit preferably includesinstructions on using the kit for detecting the nucleotide substitutionsor amino acid substitutions in TBC1D1 gene or protein, respectively.

Methods of Use Drug Screening

Polypeptides of the invention also may be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991). Thus, the invention alsoprovides a method of screening compounds to identify those that enhance(agonist) or block (antagonist) the action of TBC1D1 polypeptides orpolynucleotides, particularly those compounds potentially useful fortreating or preventing obesity and diabetes.

This invention is particularly useful for screening compounds by using awild type or mutant TBC1D1 polypeptide, or binding fragment thereof, inany of a variety of drug screening techniques. Drug screening can beperformed as described herein or using well known techniques, such asdescribed in U.S. Pat. Nos. 5,800,998 and 5,891,628, each of which areincorporated herein by reference.

The TBC1D1 polypeptide or fragment employed in such a test may either befree in solution, affixed to a solid support, or borne on a cellsurface. One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant polynucleotidesexpressing the polypeptide or fragment, preferably in competitivebinding assays. Such cells, either in viable or fixed form, can be usedfor standard binding assays. One may measure, for example, the formationof complexes between a TBC1D1 polypeptide or fragment and the agentbeing tested, or examine the degree to which the formation of a complexbetween a TBC1D1 polypeptide or fragment and a known ligand, e.g. aTBC1D1 interactor, is interfered with by the agent being tested. Forexample, because TBC1D1 has a PID domain, compounds can be screened toidentify modulators of the binding of the PID domain to phosphotyrosineor phosphotyrosine-containing proteins.

Thus, the present invention provides methods of screening test agentsfor drug candidates. Such methods comprise contacting such test agentswith a TBC1D1 polypeptide, or a fragment thereof, or a complexcontaining the TBC1D1 polypeptide, or a fragment thereof, and a bindingligand or partner protein, and assaying for the presence of a complexformed between either (a) the test agent and the TBC1D1 polypeptide orfragment, or (b) the TBC1D1 polypeptide or fragment, and the ligand orpartner protein. Methods of detecting such complexes can be any of thosewell known in the art of drug screening, but preferred methods includefluorescence polarization or fluorescent resonance energy transfer. Insuch competitive binding assays the TBC1D1 polypeptide, or a fragmentthereof, is typically labeled, and the binding ligand can beimmobilized. In assays involving competitive binding of ligands orpartner proteins, the TBC1D1 polypeptide, or fragment thereof, isallowed to reach equilibrium with a binding partner in the presence andabsence of a test compound. The free TBC1D1 polypeptide, or fragmentthereof, is then separated from that present in a TBC1D1:ligand complex,and the amount of free (i.e., uncomplexed) TBC1D1 serves as a means tomeasure the ability of the test agent to interfere with TBC1D1 ligandbinding.

Another technique used for drug screening provides high throughputscreens for compounds having suitable binding affinity to TBC1D1polypeptides and is described in detail in Geysen, PCT applicationpublication WO 84/03564. Briefly stated, large numbers of differentsmall peptide test compounds are synthesized on a solid substrate, suchas plastic pins or some other surface. The immobilized peptide testcompounds are reacted with TBC1D1 polypeptides in solution and aresubsequently washed. Bound TBC1D1 polypeptides are then detected bymethods well known in the art.

Alternatively, TBC1D1, or fragments thereof, can be immobilized on asolid phase and used for drug screening protocols. For these purposes,purified TBC1D1 can be coated directly onto plates. However,non-neutralizing antibodies to TBC1D1 can be used to capture specificantibodies to immobilize TBC1D1, or fragments thereof, on a solid phase.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of specifically bindingthe TBC1D1 polypeptide compete with a test compound for binding to theTBC1D1 polypeptide, or fragments thereof. In this manner, the antibodiescan be used to detect the presence of any peptide that shares one ormore antigenic determinants of the TBC1D1 polypeptide.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) that express awild type or mutant TBC1D1 gene and, as a consequence of expression ofwild type or mutant TBC1D1, demonstrate a specific phenotype. Thephenotype of the cells is examined to determine if the compound iscapable of modulating that phenotype, thereby indicating that thecompound affects TBC1D1 function.

Briefly, a method of screening for a substance which modulates anactivity of a polypeptide may include contacting one or more testsubstances with the polypeptide in a suitable reaction medium, testingthe activity of the treated polypeptide and comparing that activity withthe activity of the polypeptide in comparable reaction medium untreatedwith the test substance or substances. A difference in activity betweenthe treated and untreated samples indicates that the test substance orsubstances modulates the activity ot the polypeptide.

For example, it has been discovered that overexpression of TBC1D1 incertain eukaryotic cell types (e.g., Saccharomyces cerevisiae cells)causes cell death. Consequently, one method of screening for substancesthat modulate the activity of TBC1D1 would be to screen for the rescueof toxicity in cells overexpressing TBC1D1. This type of screen has beenconducted using S. cerevisiae cells and has identified several potentiallead compounds, which will be tested in human cell models, and possiblywhole animal models for obesity and/or diabetes.

Prior to, or simultaneous with, being screened for their ability tomodulate an activity of TBC1D1, test substances—especially peptides orpeptide mimetics—may be screened for their ability to directly interactwith TBC1D1 polypeptides, e.g., in a yeast two-hybrid system (e.g.,Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992;Lee et al., 1995). The yeast two-hybrid system may be used as a rapidscreen for binding activity prior to testing a substance for its abilityto modulate an activity of TBC1D1. Alternatively, a yeast two-hybridscreen could be used to screen test substances for their ability to binda TBC1D1-specific binding partner, or to act as a mimetics of a TBC1D1polypeptide.

Methods of Use Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest, or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors), in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, 1991. Rational drug designcan be performed as described herein or using well-known techniques,such as described in U.S. Pat. Nos. 5,800,998 and 5,891,628, eachincorporated herein by reference.

In one approach, one first determines the three-dimensional structure ofa protein of interest (e.g., the TBC1D1 polypeptide or fragments of theTBC1D1 polypeptide) or, perhaps, the TBC1D1-receptor or ligand complex,by x-ray crystallography, by computer modeling or most typically, by acombination of approaches. Sometimes useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson et al., 1990). Inaddition, peptides (e.g., TBC1D1 polypeptide or fragments thereof) areanalyzed by an alanine mutagenesis scan (Wells, 1991). In thistechnique, a non-alanine amino acid residue is replaced by an alanineresidue, and its effect on the peptide's activity is determined. Each ofthe amino acid residues of the peptide is analyzed in this manner todetermine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically producedpeptides. Selected peptides would then act as the pharmacore. Thus, onemay design drugs which have, e.g., improved TBC1D1 polypeptide bindingactivity or stability, or which act as inhibitors, agonists,antagonists, etc. of TBC1D1 polypeptide activity.

Following identification of a substance which modulates or affectsTBC1D1 polypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals alone or in combination with other biologically-activeagents.

Thus, the present invention extends in various aspects not only tosubstances identified as modulators of TBC1D1 activity in accordancewith what is disclosed herein, but the present invention also includespharmaceutical compositions, medicaments, drugs or other compositionscomprising such a substance, as well as methods comprisingadministration of such compositions, and methods comprisingadministration of such compositions to a patient, e.g., for treatment orprophylaxis of obesity and/or diabetes, or use of such a substance inthe manufacture of a composition for administration, e.g., for treatmentor prophylaxis of obesity and/or diabetes, and methods of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, and otheringredients as required.

A substance identified as a modulator of TBC1D1 function may be peptideor non-peptide in nature. Non-peptide “small molecules” are oftenpreferred for many in vivo pharmaceutical uses. Accordingly, a mimeticor mimic of the substance (particularly if a peptide) may be designedfor pharmaceutical use.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesize, or where it is unsuitable for a particularmethod of administration, e.g., pure peptides are unsuitable agents fororal medicaments as they tend to be quickly degraded by proteases in thealimentary canal. Mimetic design, synthesis and testing is generallyused to avoid randomly screening large numbers of molecules for a targetproperty.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. First, the particular parts ofthe compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g., by substituting each residue in turn. Alanine scans of peptidesare commonly used to refine such peptide motifs. The parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

In a variaton of this approach, the three-dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation uponbinding, allowing the model to take account of this within the design ofthe mimetic.

A template molecule is then selected onto which chemical groups thatmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, thereby protecting its ends from cellular exopeptidases. Themimetic or mimetics found by this approach can then be screened to seewhether or not they have desired properties, or to determine to whatextent they do. Further optimization or modification can then be carriedout to arrive at one or more final mimetics for testing in vivo or inclinical trials.

Methods of Use Nucleic Acid Based Therapies

According to the present invention, a method is also provided ofsupplying wild-type TBC1D1 function to a cell that carries mutant TBC1D1alleles. The wild-type TBC1D1 gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. If a gene fragment isintroduced and expressed in a cell carrying a mutant TBC1D1 allele, thegene fragment should encode a part of the TBC1D1 protein that isrequired for the normal physiological processes of the cell. Morepreferred is the situation where the wild-type TBC1D1 gene, or a partthereof, is introduced into the mutant cell in such a way that itrecombines with the endogenous mutant TBC1D1 gene present in the cell.Such recombination requires a double recombination event, which resultsin the correction of the TBC1D1 gene mutation. Vectors for theintroduction of genes, both for recombination and for extrachromosomalmaintenance, are known in the art, and any suitable vector may be used.Methods for introducing DNA into cells, such as electroporation, calciumphosphate coprecipitation and viral transduction, are also known in theart, and the choice of method is within the competence of the routineer.See also U.S. Pat. Nos. 5,800,998 and 5,891,628, each of which isincorporated by reference herein.

Among the compounds which may exhibit anti-obesity activity areantisense, ribozyme, siRNA, and triple helix molecules. Such moleculesmay be designed to reduce or inhibit mutant TBC1D1 activity. Techniquesfor the production and use of such molecules are well known to those ofskill in the art, such as described herein or in U.S. Pat. No.5,800,998, incorporated herein by reference.

Antisense RNA and DNA molecules act to either directly block thetranslation of mRNA by binding to targeted mRNA and preventing proteintranslation or by activating the cleavage of target transcripts bycellular ribonuclease-H(RNase H). With respect to antisense DNA,oligodeoxyribonucleotides directed to the translation initiation site,e.g., between the −10 and +10 regions of the TBC1D1 nucleotide sequenceof interest, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by an endonucleolytic cleavage. The composition ofribozyme molecules must include one or more sequences complementary tothe target TBC1D1 mRNA, preferably the mutant TBC1D1 mRNA, and mustinclude a well known catalytic sequence responsible for the enzymaticactivity behind mRNA cleavage. For an example of such a sequence, seeU.S. Pat. No. 5,093,246, which is incorporated by reference herein inits entirety. As such, within the scope of the invention are engineeredhammerhead motif ribozyme molecules that specifically and efficientlycatalyze the endonucleolytic cleavage of mRNA sequences encoding TBC1D1,and preferably mutant TBC1D1 proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for potentialribozyme cleavage sites which include the following sequences: GUA, GWUand GUC. Once potential cleavage sites have been identified, short RNAsequences of between 15 and 25 ribonucleotides corresponding to theregion of the target gene containing the cleavage site may be evaluatedfor predicted structural features, such as secondary structure, that mayrender the oligonucleotide sequence unsuitable for targeted cleavage byribozymes. The suitability of candidate targets may also be evaluated bytesting their accessibility to hybridization with complementaryoligonucleotides, using ribonuclease protection assays and othertechniques known in the art.

Nucleic acid molecules to be used in triplex helix formation should besingle stranded and composed of deoxynucleotides. The base compositionof these oligonucleotides must be designed to promote triple helixformation via Hoogsteen base pairing rules, which generally requiresizeable stretches of either purines or pyrimidines to be present on onestrand of a duplex. Nucleotide sequences may be pyrimidine-based, whichwill result in TAT and CGC⁺ triplets across the three associated strandsof the resulting triple helix. The pyrimidine-rich molecules providebase complementarity to a purine-rich region of a single strand of theduplex in a parallel orientation to that strand. In addition, nucleicacid molecules may be chosen that are purine-rich, for example, containa stretch of guanidine residues. These molecules will form a triplehelix with a DNA duplex that is rich in GC pairs, in which the majorityof the purine residues are located on a single strand of the targetedduplex, resulting in GGC triplets across the three strands in thetriplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with onestrand of a duplex first and then the other, eliminating the necessityfor a sizeable stretch of either purines or pyrimidines to be present onone strand of a duplex.

It is possible that the antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the translation of mRNAproduced by both normal and mutant TBC1D1 alleles. In order to ensurethat substantial normal levels of TBC1D1 activity are maintained in thecell, nucleic acid molecules that encode and express TBC1D1 polypeptidesexhibiting normal TBC1D1 activity may be introduced into cells that donot contain sequences susceptible to whatever antisense, ribozyme, ortriple helix treatments are employed. Such sequences may be introducedvia gene therapy methods. Alternatively, it may be preferable tocoadminister normal TBC1D1 protein into the cell or tissue in order tomaintain the requisite level of cellular or tissue TBC1D1 activity.

Antisense RNA and DNA molecules, ribozyme molecules and triple helixmolecules of the invention may be prepared by any method known in theart for the synthesis of DNA and RNA molecules. These include techniquesfor chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as, for example, solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro or in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

siRNAs are short intermolecular duplexes, generally composed of twodistinct (sense and antisense) strands of RNA, each of approximately 21nucleotides, that form approximately 19 base-pairs, with single stranded3′ overhangs of 1-3, preferably 2 nucleotides. The base-paired region ofsiRNAs generally substantially corresponds, preferably exactly, to a“target sequence” and its complement, in an RNA transcript to betargeted for degradation.

The specific features of siRNAs required for inducing the efficientdegradation or silencing of corresponding RNA transcripts have beensystematically investigated, as have the features of the target sequencewithin the targeted transcript. The results of such experiments havebeen published and general guideline have been established for thedesign of effective siRNA molecules (see: Tuschl et al., Genes & Dev.13:3191-3197 (1999) and Elbashir et al., EMBO J. 20:6877-6888 (2001),and discussions in “The siRNA User Guide”.

Generally, the most effective silencing is obtained with siRNA duplexescomposed of 21 nucleotide sense and antisense strands that are paired ina manner to produce 2 nucleotide 3′ overhangs. The sequence of theoverhangs makes only a small contribution to the overall specificity oftarget recognition, but the identity of the nucleotide adjacent to thepaired region can have an effect. In addition, the 3′ overhangs can becomposed either ribonucleotides or 2′-deoxyribonucleotides, with noapparent differences in efficacy, however siRNAs with2′-deoxyribonucleotide overhangs may be more resistant to cellularnucleases.

Target sequences in targeted RNA transcripts preferably have thesequence AA(19N)UU, where N=any nucleotide, but can be any contiguous 19nucleotides. Importantly, target sequences must be chosen from thesequences present in mature mRNAs, but can reside in either coding ornon-coding regions (e.g., 5′ and 3′ UTRs). Preferably the targetsequence chosen is readily “accessible,” to the siRNA, that is, notinvolved in a stable base-paired structure within the mature transcript,and not specifically bound by an RNA-binding protein. RNA foldingalgorithms, such as the “Sfold” algorithm developed by Ding and Lawrence(described in Nucleic Acids Res. 29:1034-1046 (2001)), which isincorporated by reference in its entirety) can be useful for pickingtarget sequences that have a greater likelihood of being accessible, andtherefore efficiently targeted by a corresponding siRNA, resulting indegradation of the targeted transcript and reduction in the cellularconcentration of its encoded gene product.

One example of an siRNA for use in reducing TBC1D2 expression is ansiRNA having the following structure:

siRNAs can optionally be produced within cells from precursors such assmall hairpin RNAs (shRNAs), by the action of cellular RNases.Importantly, such shRNAs can be transcribed from expression cassettesintroduced into cells, allowing for the stable silencing of targetedgenes. Details of the intracellular expression of such shRNAs can befound in U.S. Patent Application No. 2003/0148519, which is incorporatedherein in its entirety.

Various well-known modifications to nucleic acid molecules may beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include, but are not limited to, theuse of phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

Gene therapy would be carried out according to generally acceptedmethods. For example, as described in further detail in U.S. Pat. Nos.5,837,492 and 5,800,998, and the references cited therein, all of whichare incorporated by reference herein. Expression vectors in the contextof gene therapy are meant to include those constructs containingsequences sufficient to express a polynucleotide that has been clonedtherein. In viral expression vectors, the construct contains viralsequences sufficient to support packaging of the construct. If thepolynucleotide encodes an antisense polynucleotide, shRNA, or aribozyme, expression will produce the antisense polynucleotide, shRNA,or ribozyme. Thus in this context, expression does not require that aprotein product be synthesized. In addition to the polynucleotide clonedinto the expression vector, the vector also contains a promoter that isfunctional in eukaryotic cells. The cloned polynucleotide sequence isunder control of this promoter. Suitable eukaryotic promoters includethose described above. The expression vector may also include sequences,such as selectable markers and other sequences conventionally used.

Methods of Use Peptide Therapy

Peptides that have TBC1D1 activity can be supplied to cells that carrymutant or missing TBC1D1 alleles. Peptide therapy is performed asdescribed herein or using well-known techniques, such as described inU.S. Pat. Nos. 5,800,998 and 5,891,628, each of which is incorporatedherein by reference.

Protein can be produced by expression of a cDNA template sequence inbacteria, for example, using known expression vectors. Alternatively,TBC1D1 polypeptide can be extracted from TBC1D1-producing mammaliancells. In addition, the techniques of synthetic chemistry can beemployed to synthesize TBC1D1 polypeptides. Any of such techniques canprovide the preparation of the present invention that comprises theTBC1D1 protein, or some fragment thereof. Such preparations must besubstantially free of other human proteins. This is most readilyaccomplished by recombinant synthesis within a microorganism or invitro.

Active TBC1D1 molecules can be introduced into cells, either bymicroinjection, or by use of liposomes, for example. Alternatively,active TBC1D1 molecules may be taken up by cells, either actively, or bydiffusion. Extracellular application of the TBC1D1 gene product may besufficient to affect the development and or progression of obesity.Supplying cells with polypeptides with TBC1D1 activity should lead topartial reversal of the obesity and/or diabetic phenotype. Othermolecules with TBC1D1 activity (for example, peptides, drugs or organiccompounds) may also be used to effect such a reversal. Modifiedpolypeptides having substantially similar function are also used forpeptide therapy.

Alternatively, antibodies that are both specific for mutant TBC1D1 geneproduct and interfere with its activity may be used. Such antibodies maybe generated using standard techniques described herein or usingconventional techniques, such as described in U.S. Pat. Nos. 5,837,492;5,800,998 and 5,891,628, against TBC1D1 itself or against peptidescorresponding to the binding domains of TBC1D1. Such antibodies includebut are not limited to polyclonal, monoclonal, Fab fragments, F(ab′)₂fragments, single chain antibodies, chimeric antibodies, humanizedantibodies etc.

Methods of Use Transformed Hosts; Transgenic/Knockout Animals and Models

Cells and animals that carry a mutant TBC1D1 allele can be used as modelsystems in which to study and test substances that have potential astherapeutic agents. The mutant alleles may be isolated from individualswith TBC1D1 mutations, either somatic or germline, and the DNA bearingthe mutation can be introduced into the cells or animals. Alternatively,a cell line can be engineered to carry one of the mutations in theTBC1D1 allele described above, using various molecular biologicaltechniques. After a test substance is applied to the transgenic cells,the phenotype of the cell is determined. Any trait of the transformedcells can be assessed using techniques well known in the art.Transformed hosts, such as transgenic/knockout animals and models areprepared and used as described herein or using well-known techniques,such as described in U.S. Pat. Nos. 5,800,998 and 5,891,628, each ofwhich is incorporated herein by reference. For example, mutationsresulting in R125W, V228G, L392V or equivalent thereof can beincorporated into human TBC1D1 transgenes, or to the orthologous gene ofthe host.

In preferred embodiments, cell lines or transgenic animals (mice, etc.)are provided having homologous mutations in the orthologous TBC1D1 gene,which result in R125W, V228G, and/or L392V, or the equivalents thereof.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant TBC1D1 alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous, orthologous TBC1D1 gene(s) of theanimals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, 1989;Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992;Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992;Donehower et al., 1992) to produce knockout or transplacement animals. Atransplacement is similar to a knockout because the endogenous gene isreplaced, but in the case of a transplacement the replacement is byanother version of the same gene. After test substances have beenadministered to the animals, the diabetic and/or obesity phenotype isassessed. If the test substance prevents or suppresses the diabeticand/or obesity phenotype, then the test substance is a candidatetherapeutic agent for the treatment of obesity. These animal modelsprovide an extremely important testing vehicle for potential therapeuticproducts and medicaments.

In one embodiment of the invention, transgenic animals are producedwhich contain a functional transgene encoding a functional TBC1D1polypeptide or variants thereof. Transgenic animals expressing TBC1D1transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of TBC1D1. Transgenicanimals of the present invention can also be used as models for studyingindications such as obesity and/or diabetes.

In one embodiment of the invention, a TBC1D1 transgene is introducedinto a non-human host to produce a transgenic animal expressing a human,murine or other species TBC1D1 gene. The transgenic animal is producedby the integration of the transgene into the genome in a manner thatpermits the stable expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. 1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

It may be desirable to replace the endogenous TBC1D1 by homologousrecombination between the transgene or a mutant gene and the endogenousgene; or the endogenous gene may be eliminated by deletion as in thepreparation of “knock-out” animals. Typically, a TBC1D1 gene flanked bygenomic sequences is transferred by microinjection into a fertilizedegg. The microinjected eggs are implanted into a host female, and theprogeny are screened for the expression of the transgene. Transgenicanimals may be produced from the fertilized eggs from a number ofanimals including, but not limited to reptiles, amphibians, birds,mammals, and fish. Within a particularly preferred embodiment,transgenic mice are generated which overexpress wild type TBC1D1 orexpress a mutant form of the polypeptide, e.g., the equivalent of R125W,V228G, L392V mutant described above (note both R125 and L392 areconserved in the mouse TBC1D1 gene). Alternatively, the absence of aTBC1D1 in “knock-out” mice permits the study of the effects that loss ofTBC1D1 protein has on a cell in vivo. Knockout mice also provide a modelfor the development of TBC1D1-related obesity and/or diabetes.

Methods for producing knockout animals are generally described byShastry (1995, 1998) and Osterrieder and Wolf (1998). The production ofconditional knockout animals, in which the gene is active until knockedout at the desired time is generally described by Feil et al. (1996),Gagneten et al. (1997) and Lobe and Nagy (1998). Each of thesereferences is incorporated herein by reference.

As noted above, transgenic animals and cell lines derived from suchanimals may find use in certain testing experiments. In this regard,transgenic animals and cell lines capable of expressing wild type ormutant TBC1D1 may be exposed to test substances. These test substancescan be screened for the ability to alter expression of wild-type TBC1D1,or alter the expression or function of mutant TBC1D1.

Pharmaceutical Compositions and Routes of Administration

The TBC1D1 polypeptides, antibodies, peptides and nucleic acids of thepresent invention can be formulated in pharmaceutical compositions,which are prepared according to conventional pharmaceutical compoundingtechniques. See, for example, Remington's Pharmaceutical Sciences, 18thEd. (1990, Mack Publishing Co., Easton, Pa.). The composition maycontain the active agent or pharmaceutically acceptable salts of theactive agent. These compositions may comprise, in addition to one of theactive substances, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known in the art. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active ingredient. The carrier may take a wide variety of formsdepending on the form of preparation desired for a particular route ofadministration, e.g., intravenous, oral, intrathecal, epineural orparenteral.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See, forexample, PCT Application Publication WO 96/11698.

For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes account of thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cells, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be producedin the target cell, e.g. from a viral vector such as described above orin a cell based delivery system such as described in U.S. Pat. No.5,550,050 and published PCT application Nos. WO 92/19195, WO 94/25503,WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO96/40959 and WO 97/12635, designed for implantation in a patient. Thevector could be targeted to the specific cells to be treated, or itcould contain regulatory elements which are tissue or target cellspecific. The cell based delivery system is designed to be implanted ina patient's body at the desired target site and contains a codingsequence for the active agent. Alternatively, the agent could beadministered in a precursor form for conversion to the active form by anactivating agent produced in, or targeted to, the cells to be treated.See for example, EP 425,731A and WO 90/07936.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al.,1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988; Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Hogan et al., Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Ageneral discussion of techniques and materials for human gene mapping,including mapping of human chromosome 1, is provided in White andLalouel (1988).

EXAMPLES

The present invention is described by reference to the followingExample, which is offered by way of illustration and is not intended tolimit the invention in any manner. Standard techniques well known in theart or the techniques specifically described below were utilized.

Example 1 Association of TBC1D1 and Obesity

We have conducted a genome wide search with 628 markers usingmultigenerational Utah pedigrees to identify genes involved in obesitypredisposition. Models used in this analysis were females-only andaffecteds-only with dominant, codominant and recessive modes ofinheritance. The resource used to define this linkage was 37 Utahpedigrees with 994 individuals.

From the genome search, we identified a highly significant linkage tohigh BMI at D4S2632 with a multipoint HLOD score of 6.1 (p-value10⁻⁷)and a nonparametric linkage score of 5.3 (p-value 10⁻⁶). To pursue the4p14-15 linkage, we increased both the marker density around D4S2632 andthe size of our pedigree data set. As a result, the linkage evidenceincreased to a HLOD score of 9.2 (at D4S3350, p-value 10⁻¹⁰) and anonparametric linkage score of 11.3 (p-value 10⁻¹²). The fraction offamilies that support the linkage (α) is 0.46. The region defined by thelinkage evidence is 10 cM or about 10 million bases and containsapproximately 20 genes.

From fifteen families with good evidence for linkage to this region, twopedigree members that shared the segregating haplotype were mutationscreened. Variants that change the encoded amino acid (missensechanges), were scrutinized for evidence that the variant changes thefunction of the gene and that the change results in a higher risk ofobesity. A gene carrying such variants would be a good candidate for theobesity susceptibility gene. Causal variants will segregate into otheraffected individuals in the family and will be more rare than innon-cases than in cases.

The TBC1D1 consensus cDNA sequence is 3791 nucleotides and is covered bythree human genomic DNA sequences (GenBank accessions AC021106,AC009595, and AC044902). It has a 1168-amino acid open reading framewith an initiating methionine codon (ATG) at nucleotides 1-3 and a 3′STOP codon (TAG) at nucleotides 3505-3507. The amino acid sequence hasan 95% identity to the mouse Tbc1 protein (GenBank accession numberT29104). Both the initiation codon and the 3′ STOP codon are conserved.

The TBC1D1 cDNA sequence was constructed using the mouse tbc1 mRNAsequence (GenBank accession U33005), as a template and assembling humansequence available in the public databases. The majority of the TBC1D1cDNA sequence, nucleotide positions 1216 to 3791, is derived from apartial coding sequence, KIAA1108 (GenBank accession AB029031). A seriesof three overlapping ESTs (GenBank accession numbers AI872406, AW204569,and BE279997) were assembled to generate the 5′ end of TBC1D1,nucleotide positions 1-1491.

TBC1D1 is the founding member of a family of related proteins withhomology to tre-2/UPS6, BUB2, and cdc16 and containing the TBC box motifat amino acids 180-220. In mice, Tbc1 showed differential expression intwo mast cell lines. It was localized in the nucleus, and was expressedat the highest levels in hematopoietic cells, testis and kidney. Withinthese tissues, expression of Tbc1 was cell- and stage-specific. Based onsequence similarity, pattern of expression and subcellular localization,Tbc1 may play a role in the cell cycle and in the differentiation ofvarious tissues.

Two missense changes (see Table 1) were detected. Arg→Trp (R125W) infamilies 436, 7082, 7380, 7228, 7256.1, 11063, 7158.2 and 1220. Thisvariant was enriched in our linked-cases (p value 10⁻⁴) as compared torandom controls, and segregated with obesity in 6 families. R125 isconserved in mice and is in the phosphotyrosine interacting domain(PID). We have also detected a gene haplotype containing both Val→Glyand Leu→Val, which segregates with the disease in families 736201, 7444,7390, and 740701. This haplotype is also enriched in linked cases ascompared to random controls. Individually, the variants are enriched inlinked-cases with p-values of 0.02 and 0.01 respectively.

TABLE 1 Alterations in TBC1D1 Associated with Obesity Observa- p-valueNucleo- Amino tions in for Linked- tide Acid control chro- cases vs.Kindred Variant Change Change mosomes Controls 436 Arg → Trp C373T R125W 22/334 0.0001 7082 Arg → Trp C373T R125W 7380 Arg → Trp C373T R125W7228 Arg → Trp C373T R125W 7256.1 Arg → Trp C373T R125W 11063 Arg → TrpC373T R125W 7158.2 Arg → Trp C373T R125W 1220 Arg → Trp C373T R125W736201 Val → Gly; T683G; V228G; 119/340; 0.02; Leu → Val C1174G L392V 40/350 0.01 7444 Val → Gly; T683G; V228G; Leu → Val C1174G L392V 7390Val → Gly; T683G; V228G; Leu → Val C1174G L392V 74701 Val → Gly; T683G;V228G; Leu → Val C1174G L392V Shaded boxes: variant segregates withobesity in indicated family

Example 2 Cell and Animal Obesity Disease Models

Compounds identified by the drug screens of the invention (i.e., thosethat modify TCB1D1 bioactivity) can be further tested in obesity diseasemodels. Preferably, any compound or molecule identified as being capableof affecting the bioactivity of TCB1D1 in a primary screen is tested inan animal or cell-based obesity disease model. Compounds that showactivity in the secondary screen (i.e., obesity disease model) areidentified as having obesity disease modifying activity.

The skilled artisan is capable of testing compounds that affect TCB1D1bioactivity in obesity disease models. Any obesity disease model can beused. Preferably, drug candidates that have obesity disease modifyingactivity, show an effect in several different obesity disease models. Itis preferred that the drug candidates that have obesity diseasemodifying activity show a disease modifying affect in at least 2, 3, 4,or more obesity disease models.

A variety of obesity disease models are known to the skilled artisan. Apreferred disease models is a transgenic animal, preferably a mouse orrat that has an altered TCB1D1 gene variant associated with obesity. Theanimal can be heterozygous or homozygous for said TCB1D1 variant.Preferably the animal displays an observable phenotype (e.g., is obese)that is detectably different from the wild-type phenotype. Drugcandidates identified as affecting TCB1D1 bioactivity in the primaryscreen are then tested in a group of transgenic animals. A drugcandidate having obesity disease modifying activity decreases obesityassociated with the transgenic animal having the TCB1D1 variants ascompared to a group of control transgenic animal that are not treatedwith the drug candidate. Similarly other disease models can be used suchas obese mice, obese rats, and transgenic animals that have an obesephenotype. See, e.g., Chagnon et al. Obesity Res. 11:313-267 (2003);Tran et al. Surg. 134:372-377 (2003); Farley et al. Obesity Res.11:845-851 (2003); Dobrian et al. Am J. Physiol. Renal Physiol. (Jun. 102003 Epub); Kero et al. Am. J. Physiol. Endo. Metab. (May 28, 2003Epub); Serra et al. J. Cell. Physiol. 196:89-97 (2003); Bailhache et al.Metabolism 52:559-564 (2003); Masaki et al. Endocrinology 144:2741-2748(2003); Uehara et al. Int. J. Mol. Med. 11:723-727 (2003); Woods et al.J. Nutr. 133:1081-1087 (2003); Miyasaka et al. Mech. Ageing Dev.124:183-190 (2003); and Comuzzie et al. Obesity Res. 11:75-80 (2003).

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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1. A method for determining that a human subject is at risk fordeveloping obesity comprising: assaying a sample from a human subject,said sample comprising a TBC1D1-encoding nucleic acid molecule or thecomplement thereof, detecting a cytidine to thymidine alteration at the373^(rd) nucleotide of the TBC1D1 coding sequence of SEQ ID NO:1,wherein the presence of said cytidine to thymidine alteration identifiesthe subject as being at risk for developing obesity.
 2. The method ofclaim 1 wherein said assaying step is conducted on genomic DNA.
 3. Themethod of claim 1 wherein said assaying step is conducted on mRNA. 4.The method of claim 1, wherein said cytidine to thymidine alteration isdetected by a method selected from the group consisting of: a)hybridizing a probe specific for said alteration to RNA isolated fromsaid human sample and detecting the presence of a hybridization product,wherein the presence of said product indicates the presence of saidalteration in the sample; b) hybridizing a probe specific for saidalteration to cDNA made from RNA isolated from said sample and detectingthe presence of a hybridization product, wherein the presence of saidproduct indicates the presence of said alteration in the sample; c)hybridizing a probe specific for said alteration to genomic DNA isolatedfrom said sample and detecting the presence of a hybridization product,wherein the presence of said product indicates the presence of saidalteration in the sample; d) amplifying all or part of saidTBC1D1-encoding nucleic acid molecule, or complement thereof, in saidsample using a set of primers to produce amplified nucleic acids andsequencing the amplified nucleic acids; e) amplifying part of saidTBC1D1-encoding nucleic acid molecule, or complement thereof, in saidsample using a primer specific for said alteration and detecting thepresence of an amplified product, wherein the presence of said productindicates the presence of said alteration in the sample; f) molecularlycloning all or part of said TBC1D1-encoding nucleic acid molecule, orcomplement thereof, in said sample to produce a cloned nucleic acid andsequencing the cloned nucleic acid; g) amplifying said TBC1D1-encodingnucleic acid molecule, or complement thereof, to produce amplifiednucleic acids, hybridizing the amplified nucleic acids to a DNA probespecific said alteration and detecting the presence of a hybridizationproduct, wherein the presence of said product indicates the presence ofsaid alteration; h) forming single-stranded DNA from a gene fragment ofsaid TBC1D1-encoding nucleic acid molecule, or complement thereof, fromsaid human sample and single-stranded DNA from a corresponding fragmentof a wild-type gene, electrophoresing said single-stranded DNAs on anon-denaturing polyacrylamide gel and comparing the mobility of saidsingle-stranded DNAs on said gel to determine if said single-strandedDNA from said sample is shifted relative to wild-type and sequencingsaid single-stranded DNA having a shift in mobility; i) forming aheteroduplex consisting of a first strand of nucleic acid selected fromthe group consisting of a genomic DNA fragment isolated from saidsample, an RNA fragment isolated from said sample and a cDNA fragmentmade from mRNA from said sample and a second strand of a nucleic acidconsisting of a corresponding human wild-type gene fragment, analyzingfor the presence of a mismatch in said heteroduplex, and sequencing saidfirst strand of nucleic acid having a mismatch; j) formingsingle-stranded DNA from said TBC1D1-encoding nucleic acid molecule, orcomplement thereof, of said human sample and from a correspondingfragment of an allele specific for said alteration, electrophoresingsaid single-stranded DNAs on a non-denaturing polyacrylamide gel andcomparing the mobility of said single-stranded DNAs on said gel todetermine if said single-stranded DNA from said sample is shiftedrelative to said allele, wherein no shift in electrophoretic mobility ofthe single-stranded DNA relative to the allele indicates the presence ofsaid alteration in said sample; and k) forming a heteroduplex consistingof a first strand of nucleic acid selected from the group consisting ofa genomic DNA fragment of said TBC1D1-encoding nucleic acid molecule, orcomplement thereof, isolated from said sample, an RNA fragment isolatedfrom said sample and a cDNA fragment made from mRNA from said sample anda second strand of a nucleic acid consisting of a corresponding geneallele fragment specific for said alteration and analyzing for thepresence of a mismatch in said heteroduplex, wherein no mismatchindicates the presence of said alteration.
 5. The method of claim 1,wherein said assaying step comprises hybridizing a nucleic acid probespecifically hybridizable to an altered TBC1D1 coding sequence orcomplement thereof.
 6. A method for predicting, in a human subject, thelikelihood of developing obesity associated with a genetic variant ofthe human TBC1D1 gene comprising: detecting the presence of a cytidineto thymidine alteration at the 373^(rd) nucleotide of the TBC1D1 codingsequence of SEQ ID NO:1, wherein the presence of said cytidine tothymidine alteration predicts that said subject has an increasedlikelihood of developing obesity.
 7. The method of claim 6, wherein saidcytidine to thymidine alteration is detected by determining the genomicsequence of said TBC1D1 gene.